Generation of neural stem cells and motor neurons

ABSTRACT

A method of generating a population of cells useful for treating a brain disorder in a subject is disclosed. The method comprises contacting mesenchymal stem cells (MSCs) with at least one exogenous miRNA having a nucleic acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 15-19 and 27-35, thereby generating a population of cells and/or generating neurotrophic factors that may provide important signals to damaged tissues or locally residing stem cells. MSCs differentiated by miRs may also secrete miRs and deliver them to adjacent cells and therefore provide important signals to neighboring endogenous normal or malignant cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. patent applicationSer. No. 14/380,165 filed on Aug. 21, 2014 (now U.S. Pat. No. 9,803,175)and titled “Generation of Neural Stem Cells and Motor Neurons”, which isa national phase of PCT Patent Application No. PCT/IB2013/051429 filedon Feb. 21, 2013, which claims the benefit of priority of U.S.Provisional Patent Application No. 61/601,596 filed on Feb. 22, 2012.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof ex vivo differentiating mesenchymal stem cells towards neural stemcells and motor neurons using microRNAs (miRNAs).

Mesenchymal stem cells (MSCs) are a heterogeneous population of stromalcells that can be isolated from multiple species, residing in mostconnective tissues including bone marrow, adipose, placenta, umbilicalcord and perivascular tissues. MSCs can also be isolated from theplacenta, amniotic fluid and cord's Wharton's jelly.

The concentration of MSCs in all tissues, including bone marrow andadipose tissue is very low but their number can be expanded in vitro.Typically, expansion of MSCs using up to 15 passages does not result inmutations indicating genetic stability. MSC can differentiate into cellsof the mesenchymal lineage, such as bone, cartilage and fat but, undercertain conditions, have been reported to acquire the phenotype of cellsof the endodermal and neuroectodermal lineage, suggesting some potentialfor “trans-differentiation”.

Within the bone marrow compartment, these cells are tightly intermingledwith and support hematopoiesis and the survival of hematopoietic stemcells in acquiescent state (7). In addition, after expansion in culture,MSCs retain their ability to modulate innate and adaptive immunity (8).Furthermore, MSCs migrate actively to sites of inflammation and protectdamaged tissues, including the CNS, properties that supported their useas new immunosuppressive or rather immunoregulatory or anti-inflammatoryagents for the treatment of inflammatory and immune-mediated diseasesincluding autoimmune disorders (9). These features of MSCs merited theiruse to control life-threatening graft-versus-host-disease (GVHD)following allogeneic bone marrow transplantation, thus controlling oneof the most serious complications of allogenic bone marrowtransplantation, helping to lower transplant-related toxicity andmortality associated with multi-system organ injury (10).

Several studies have shown that MSCs following exposure to differentfactors in vitro, change their phenotype and demonstrate neuronal andglial markers [Kopen, G. C., et al., Proc Natl Acad USA. 96(19):10711-6,1999; Sanchez-Ramos, et al. Exp Neurol. 164(2):247-56. 2000; Woodbury,D., J Neurosci Res. 61(4):364-70,2000; Woodbury, D., et al., J NeurosciRes. 69(6):908-17, 2002; Black, I. B., Woodbury, D. Blood Cells Mol Dis.27(3):632-6, 2001; Kohyama, J., et al. Differentiation. 68(4-5):235-44,2001; Levy, Y. S. J Mol Neurosci. 21(2):121-32, 2003].

Accordingly, MSCs (both ex-vivo differentiated and non-differentiated)have been proposed as candidates for cell replacement therapy for thetreatment of various neurological disorders including multiplesclerosis, Parkinson's disease, ALS, Alzheimer's disease, spinal cordinjury and stroke.

Motor neurons in the spinal cord innervate skeletal muscles, andoriginate from neuroepithelial cells in a restricted area of thedeveloping spinal cord (neural tube). During embryonic development,motor neurons extend their processes (nerves) to the periphery toinnervate skeletal muscles that are adjacent to the spinal cord. In anadult human body, however, motor neuron's axons are projected largedistances away from the cell bodies in the spinal cord to reach theirtarget muscles. Because of this, motor neurons have a higher metabolicrate compared to smaller neurons, and this renders them more susceptibleto genetic, epigenetic, and environmental changes. Motor neurons cannotrenew themselves and therefore their loss or degeneration are generallyassociated with fatal neurological conditions including paralysis anddisorders such as pediatric spinal muscular atrophy (SMA) and adultonset amyotrophic lateral sclerosis (ALS).

Roy et al., 2005 [Exp Neural. 2005; 196:224-234]; Zhang et al., 2006[Stem Cells. 2006; 24:434-442]; Bohl et al., 2008 [Stem Cells. 2008;26:2564-2575]; and Dimos et al., 2008 [Science. 2008; 321:1218-1221] thecontents of which are incorporated by reference teach geneticmodification of different stem cells to induce differentiation intomotor neurons.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of predisposing mesenchymal stem cells todifferentiate into neural stem cells, the method comprisingup-regulating a level of at least one exogenous miRNA selected from thegroup consisting of miR-1275, miR-891a, miR-154, miR-1202, miR-572,miR-935a, miR302b, miR-371, miR-134, miR-219, miR-155, miR-32, miR-33,miR-126, miR-127, miR-132, let-7c, miR-665, miR-4258, miR-361-3p,miR-374a-star, miR-892b, miR-361-5p, miR-181a, miR-16, miR-636,miR-4284, miR-1208, miR-1274b, miR-30c-2-star, miR-501-3p, hsa-miR-92a,miR-378b, miR-1287, miR-425-star, miR-324-5p, miR-3178, miR-219-1-3p,miR-197, miR-181b, miR-500-star, miR-106b, miR-502-3p, miR-30c,miR-1275, miR-422a, miR-93, miR-181d, miR-1307, miR-1301, miR-99a,miR-505-star, miR-1202, miR-12, miR-532-5p, miR-195, miR-532-3p,miR-106a, miR-17, miR-1271, miR-769-3p, miR-15b, miR-324-3p, miR-20a,miR-501-5p, miR-330-3p, miR-874, miR-500, miR-25, miR-769-5p,miR-125b-2-star, miR-130b, miR-504, miR-181a-2-star, miR-885-3p,miR-1246, miR-92b, miR-362-5p, miR-572, miR-4270, miR-378c, miR-93-star,miR-149, miR-363, miR-9, miR-18a, miR-346, miR-497, miR-378, miR-1231,miR-139-5p, miR-3180-3p, miR-935 and miR-20b in the mesenchymal stemcells (MSCs), thereby predisposing the MSCs to differentiate into theneural stem cells.

According to an aspect of some embodiments of the present inventionthere is provided a method of predisposing MSCs to differentiate intoneural stem cells, the method comprising down-regulating an expressionof at least one miRNA selected from the group consisting of miR-4317,miR-153, miR-4288, miR-409-5p, miR-193a-5p, miR-1Ob, miR-142-3p,miR-131a, miR-125b, miR-181a, miR-145, miR-143, miR-214, miR-199a-3p,miR-199a-5p, miR-199b-3p, miR-138, miR-31, miR-21, miR-193a-5p,miR-224-star, miR-196a, miR-487b, miR-409-5p, miR-193b-star, miR-379,miR-21-star, miR-27a-star, miR-27a, miR-4317, miR-193b, miR-27b, miR-22,574-3p, miR-4288, miR-23a, miR-221-star, miR-2113, let-7i, miR-24,miR-23b, miR-299-3p, miR-518c-star, miR-221, miR-431-star, miR-523,miR-4313, miR-559, miR-614, miR-653, miR-2278, miR-768-5p, miR-154-star,miR-302a-star, miR-3199 and miR-3137 in the mesenchymal stem cells byup-regulating a level of at least one polynucleotide agent thathybridizes and inhibits a function of the at least one miRNA therebypredisposing the MSCs to differentiate into the neural stem cells.

According to an aspect of some embodiments of the present inventionthere is provided a method of predisposing MSCs to differentiate intoneural stem cells, the method comprising up-regulating a level ofexogenous miR-124 in the mesenchymal stem cells (MSCs) anddown-regulating a level of miR-let-7 in the MSCs, thereby predisposingthe MSCs to differentiate into the neural stem cells.

According to an aspect of some embodiments of the present inventionthere is provided a method of predisposing MSCs to differentiate intoneural stem cells, the method comprising contacting the mesenchymal stemcells (MSCs) with an agent that down-regulates an amount and/or activityof Related to testis-specific, vespid and pathogenesis protein 1(RTVP-1), thereby predisposing MSCs to differentiate into the neuralstem cells.

According to an aspect of some embodiments of the present inventionthere is provided a method of predisposing neural stem cells todifferentiate into motor neurons, the method comprising up-regulating alevel of at least one exogenous miRNA selected from the group consistingof miR-368, miR-302b, miR-365-3p, miR-365-5p, miR-Let-7a, miR-Let-7b,miR-218, miR-134, miR-124, miR-125a, miR-9, miR-154, miR-20a andmiR-130a in neural stem cells (NSCs), thereby predisposing NSCs todifferentiate into the motor neurons.

According to an aspect of some embodiments of the present inventionthere is provided a method of predisposing MSCs to differentiate intomotor neurons, the method comprising up-regulating a level of at leastone exogenous miRNA selected from the group consisting of miR-648,miR-368, miR-365, miR-500, miR-491, miR-218, miR-155, miR-192, let-7b,miR-16, miR-210, miR-197, miR-21, miR-373, miR-27a, miR-122, miR-17,miR-494, miR-449, miR-503, miR-30a, miR-196a, miR-122, miR-7,miR-151-5p, miR-16, miR-22, miR-31, miR-424, miR-1, miR-29c, miR-942,miR-100, miR-520, miR-663a, miR-562, miR-449a, miR-449b-5p, miR-520b,miR-451, miR-532-59, miR-605, miR-504, miR-503, miR-155, miR-34a,miR-16, miR-7b, miR-103, miR-124, miR-1385p, miR-16, miR-330, miR-520,miR-608, miR-708, miR-107, miR-137, miR-132, miR-145, miR-204, miR-125b,miR-224, miR-30a, miR-375, miR-101, miR-106b, miR-128, miR-129-5p,miR-153, miR-203, miR-214, miR-338-3p, miR-346, miR-98, miR-107,miR-141, miR-217, miR-424, miR-449, miR-7, miR-9, miR-93, miR-99a,miR-100, miR-1228, miR-183, miR-185, miR-190, miR-522, miR-650, miR-675,miR-342-3p, miR-31 in the mesenchymal stem cells (MSCs), therebypredisposing MSCs to differentiate into the motor neurons.

According to an aspect of some embodiments of the present inventionthere is provided a method of predisposing NSCs to differentiate intomotor neurons, the method comprising down-regulating an expression of atleast one miRNA selected from the group consisting of miR-372, miR-373,miR-141, miR-199a, miR-32, miR-33, miR-221 and miR-223 by up-regulatinga level of at least one polynucleotide agent that hybridizes andinhibits a function of the at least one miRNA in the NSCs therebypredisposing NSCs to differentiate into the motor neurons.

According to an aspect of some embodiments of the present inventionthere is provided a method of predisposing MSCs to differentiate intomotor neurons, the method comprising down-regulating an expression of atleast one miRNA selected from the group consisting of miR-372, miR-373,miR-942, miR-2113, miR-199a-3p, miR-199a-5p, miR-372, miR-373, miR-942,miR-2113, miR-301a-3p, miR-302c, miR-30b-5p, miR-30c, miR-326, miR-328,miR-331-3p, miR-340, miR-345, miR-361-5p, miR-363, miR-365a-3p,miR-371a-3p, miR-373-3p, miR-374a, miR-423-3p, miR-449b-5p, miR-451a,miR-494, miR-504, miR-515-3p, miR-516a-3p, miR-519e, miR-520a-3p,miR-520c-3p, miR-520g, miR-532-5p, miR-559, miR-562, miR-572,miR-590-5p, miR-605, miR-608, miR-626, miR-639, miR-654-3p, miR-657,miR-661, miR-708-5p, miR-942, miR-96, miR-99amo and miR-194 byup-regulating a level of at least one polynucleotide agent thathybridizes and inhibits a function of the at least one miRNA in the MSCsthereby predisposing MSCs to differentiate into the motor neurons.

According to an aspect of some embodiments of the present inventionthere is provided a genetically modified isolated population of cellswhich comprise at least one exogenous miRNA selected from the groupconsisting of miR302b, miR-371, miR-134, miR-219, miR-154, miR-155,miR-32, miR-33, miR-126, miR-127, miR-132 and miR-137 and/or whichcomprise at least one polynucleotide agent that hybridizes and inhibitsa function of at least one miRNA selected from the group consisting ofmiR-10b, miR-142-3p, miR-131a, miR-125b, miR-153 and miR-181a, whereinthe cells have a neural stem cell phenotype.

According to an aspect of some embodiments of the present inventionthere is provided a genetically modified isolated population of cellswhich comprise at least one exogenous miRNA selected from the groupconsisting of miR-368, miR-302b, miR-365-3p, miR-365-5p, miR-Let-7a,miR-Let-7b, miR-218, miR-134, miR-124, miR-125a, miR-9, miR-154,miR-20a, miR-130a and/or which comprise at least one polynucleotideagent that hybridizes and inhibits a function of at least one miRNAselected from the group consisting of miR-372, miR-373, miR-141,miR-199a, miR-32, miR-33, miR-221 and miR-223, wherein the cells have amotor neuron phenotype.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a brain disease or disorder in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of the isolated population ofcells of claim 33, thereby treating the brain disease or disorder.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the isolatedpopulation of cells described herein and a pharmaceutically acceptablecarrier.

According to an aspect of some embodiments of the present inventionthere is provided a method of selecting a miRNA which may be regulatedfor the treatment of a motor neuron disease comprising:

(a) differentiating a population of neural stem cells towards a motorneuron phenotype; and

(b) analyzing a change in expression of a miRNA in the population ofMSCs prior to and following the differentiating of the MSCs towards amotor neuron phenotype, wherein a change of expression of a miRNA aboveor below a predetermined level is indicative that the miRNA may beregulated for the treatment of the motor neuron disease.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a motor neuron disease in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of the isolated population ofcells of claim 35, thereby treating the brain disease or disorder.

According to an aspect of some embodiments of the present inventionthere is provided a genetically modified isolated population of cellswhich comprise at least one exogenous miRNA selected from the groupconsisting of miR-1275, miR-891a, miR-154, miR-1202, miR-572 andmiR-935a and/or which comprise at least one polynucleotide agent thathybridizes and inhibits a function of at least one miRNA selected fromthe group consisting of miR-4317, miR-153, miR-4288, miR-409-5p,miR-193a-5p, wherein said cells have a neural stem cell phenotype.

According to an aspect of some embodiments of the present inventionthere is provided a genetically modified isolated population of cellswhich comprise at least one exogenous miRNA selected from the groupconsisting of miR-648, miR-368, miR-365, miR-500 and miR-491 and/orwhich comprise at least one polynucleotide agent that hybridizes andinhibits a function of at least one miRNA selected from the groupconsisting of miR-372, miR-373, miR-942, miR-2113, miR-199a-3p andmiR-199a-5p, wherein said cells have a motor neuron phenotype.

According to some embodiments of the invention, the at least oneexogenous miRNA is selected from the group consisting of miR-1275,miR-891a, miR-154, miR-1202, miR-572 and miR-935a.

According to some embodiments of the invention, the at least oneexogenous miRNA is selected from the group consisting of miR-20b,miR-925, miR-891 and miR-378.

According to some embodiments of the invention, the at least one miRNAis selected from the group consisting of miR-4317, miR-153, miR-4288,miR-409-5p, and miR-193a-5p.

According to some embodiments of the invention, the at least one miRNAis selected from the group consisting of miR-138, miR-214, miR-199a andmiR-199b.

According to some embodiments of the invention, the at least one miRNAis miR-138, the method further comprises:

(i) down-regulating an expression of miR-891 using a polynucleotideagent that hybridizes and inhibits the function of miR-891;

(ii) up-regulating a level of exogenous miR20b; or

(iii) up-regulating a level of exogenous miR378.

According to some embodiments of the invention, the miRNA is selectedfrom the group consisting of miR-648, miR-368, miR-365, miR-500 andmiR-491.

According to some embodiments of the invention, the miRNA is selectedfrom the group consisting of miR-372, miR-373, miR-942, miR-2113,miR-199a-3p and miR-199a-5p.

According to some embodiments of the invention, the at least one miRNAcomprises each of miR Let-7a, miR-124, miR-368 and miR-154.

According to some embodiments of the invention, the at least one miRNAcomprises each of miR-125a, miR-9 and miR-130a.

According to some embodiments of the invention, the at least one miRNAcomprises each of miR-218, miR-134 and miR-20a.

According to some embodiments of the invention, the method furthercomprises down-regulating each of miR-141, miR-32, miR-33, miR-221,miR-223 and miR-373.

According to some embodiments of the invention, the NSCs are generatedby ex vivo differentiating MSCs.

According to some embodiments of the invention, the ex vivodifferentiating is affected according to any of the methods describedherein.

According to some embodiments of the invention, the MSCs are isolatedfrom a tissue selected from the group consisting of bone marrow, adiposetissue, placenta, cord blood and umbilical cord.

According to some embodiments of the invention, the MSCs are autologousto the subject.

According to some embodiments of the invention, the MSCs arenon-autologous to the subject.

According to some embodiments of the invention, the MSCs aresemi-allogeneic to the subject.

According to some embodiments of the invention, the up-regulatingcomprises introducing into the MSCs the at least one miRNA.

According to some embodiments of the invention, the up-regulating isaffected by transfecting the MSCs with an expression vector whichcomprises a polynucleotide sequence which encodes a pre-miRNA of the atleast one miRNA.

According to some embodiments of the invention, the up-regulating isaffected by transfecting the MSCs with an expression vector whichcomprises a polynucleotide sequence which encodes the at least onemiRNA.

According to some embodiments of the invention, the method furthercomprises analyzing an expression of at least one marker selected fromthe group consisting of nestin and Sox2 following the generating.

According to some embodiments of the invention, the method furthercomprises analyzing an expression of at least one marker selected fromthe group consisting of Islet1, HB9 and the neuronal markersneurofilament and tubulin following the generating.

According to some embodiments of the invention, the method is effectedin vivo. According to some embodiments of the invention, the method iseffected ex vivo.

According to some embodiments of the invention, at least 50% of thepopulation of cells express at least one marker selected from the groupconsisting of nestin and Sox2.

According to some embodiments of the invention, the at least 50% of thepopulation of cells express at least one marker selected from the groupconsisting of Islet1, HB9 and the neuronal markers neurofilament andtubulin.

According to some embodiments of the invention, the isolated populationof cells is for use in treating a brain disease or disorder.

According to some embodiments of the invention, the isolated populationof cells is for brain disease or disorder is a neurodegenerativedisorder.

According to some embodiments of the invention, the neurodegenerativedisorder is selected from the group consisting of multiple sclerosis,Parkinson's, epilepsy, amyotrophic lateral sclerosis (ALS), stroke, RettSyndrome, autoimmune encephalomyelitis, spinal cord injury, cerebralpalsy, stroke, Alzheimer's disease and Huntingdon's disease.

According to some embodiments of the invention, the isolated populationis for use in treating a motor neuron disease.

According to some embodiments of the invention, the motor neuron diseaseis selected from the group consisting of amyotrophic lateral sclerosis(ALS), primary lateral sclerosis (PLS), pseudobulbar palsy andprogressive bulbar palsy.

According to some embodiments of the invention, the nerve disease ordisorder is a neurodegenerative disorder.

According to some embodiments of the invention, the neurodegenerativedisorder is selected from the group consisting of multiple sclerosis,Parkinson's, epilepsy, amyotrophic lateral sclerosis (ALS), stroke, RettSyndrome, autoimmune encephalomyelitis, spinal cord injury, cerebralpalsy, stroke, Alzheimer's disease and Huntingdon's disease.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings and images.With specific reference now to the drawings in detail, it is stressedthat the particulars shown are by way of example and for purposes ofillustrative discussion of embodiments of the invention. In this regard,the description taken with the drawings makes apparent to those skilledin the art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-B are photographs and graphs illustrating that mesenchymal stemcells (MSCs) may be induced to differentiate to neural stem cell(NSC)-like cells and express NSC markers. MSCs were plated inneurosphere medium on bacteria dishes as described in Methods.

The MSC-derived spheroids were characterized by immunofluorescence (FIG.1A) and real-time PCR (FIG. 1 B).

FIG. 2 is a bar graph illustrating exemplary miRNAs associated with stemcell signature and self renewal that were up-regulated during NSCdifferentiation.

FIG. 3 is a bar graph illustrating exemplary miRNAs associated withhematopoiesis that were up-regulated during NSC differentiation.

FIGS. 4A-D are bar graphs illustrating exemplary miRNAs associated witha neuronal signature and self renewal that were up-regulated (FIGS.4A-C) or down-regulated (FIG. 4D) during NSC differentiation.

FIGS. 4E-F are photographs illustrating bone marrow MSCs transfectedwith antagomiR-138 and miR-891 using a nestin promoter reporter assay.

FIGS. 5A-D are graphs and photographs illustrating that RTVP-1 plays arole in differentiation of MSCs towards NSCs. RTVP-1 is expressed inhigh levels in BM-MSCs, similar to some glioma cells that are consideredas the cells that expressed the highest levels of this protein, asdetermined by Western blot analysis (A). A diagram showing themesenchymal lineage differentiation of MSCs (B). Silencing of RTVP-1 inBM-MSCs using siRNA duplexes decreases the osteogenic differentiation ofthese cells (C). Silencing of RTVP-1 in BM-MSCs decreases the expressionof the different mesenchymal markers (D).

FIG. 5E is a bar graph illustrating the expression of RTVP-1 in MSCs andMSCs differentiated to NSCs.

FIG. 5F is a bar graph illustrating the effect of silencing of RTVP-1 onnestin expression in MSCs.

FIGS. 6A-D are photographs and graphs illustrating the effect oftransfection of Olig2 and differentiation medium on placenta-derivedMSCs. After 12 days in culture the cells were analyzed for theexpression of motor neuron progenitor (FIG. 6C) and motor neuron markers(FIG. 6D) using real time PCR. FIG. 6A illustrates undifferentiatedMSCs. FIG. 6B illustrates differentiated MSCs.

FIGS. 7A-B are graphs and photographs illustrating that NSCs may beinduced to differentiate into motor neuron cells. The human neuralprogenitor cells (Lonza) were grown as spheroids and then plated onlaminin and treated with the different factors as described in themethods. Following 12-14 days, the cells were analyzed for morphologicalappearance and for the different markers using real time PCR.

FIG. 8 is a bar graph illustrating exemplary miRNAs associated with stemcell signature and self renewal that were up-regulated during motorneuron differentiation.

FIG. 9 is a bar graph illustrating exemplary miRNAs associated withhematopoiesis that were up-regulated during motor neurondifferentiation.

FIG. 10 is a bar graph illustrating exemplary miRNAs associated with aneuronal signature and self-renewal that were up-regulated during motorneuron differentiation.

FIG. 11 is a bar graph illustrating Islet1 and HB9 mRNA expression incontrol MSCs and MSCs trans-differentiated toward a motor neuron cell.

FIG. 12 is a bar graph illustrating nestin mRNA expression in controlMSCs, MSCs with RTVP-1 silencing, and MSCs with RTVP-1 silencing andmiR/antimiR transfection.

FIG. 13 is a bar graph illustrating the average score on the Basso,Beattie and Bresnahan (BBB) locomotor scale of rats with and withoutspinal cord injury and with injury treated with MSCstrans-differentiated toward a motor neuron cell.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof ex vivo differentiating mesenchymal stem cells towards neuralprogenitor cells and motor neurons using microRNAs.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Neural stem cells (NSCs) have been isolated from embryonic and fetalmammalian and human brains and propagated in vitro in a variety ofculture systems (Doetsch et al., 1999, Subventricular zone astrocytesare neural stem cells in the adult mammalian brain. Cell 97:703-16,Johansson et al., 1999, Cell 96:25-34, Svendsen et al., 1998, J NeurosciMethods 85:141-52). A system for proliferating human neural stem cells(hNSCs) in serum-free culture medium containing human bFGF and human EGFalso has been reported (Kim et al., 2002, Proc Natl Acad Sci USA 99:4020-4025, Qu et al., 2001, NeuroReport 12: 1127-1132). Further,transplantation of hNSCs into experimental animals has been described(Qu et al., 2001, Id.; Qu et al., 2005, 35^(th) Annual Meeting inWashington, D.C., November 2005).

However, challenges existed in the art of stem cell therapies using stemcells derived from embryonic/fetal tissue sources. Stem cell therapiesusing embryonic sources face challenges such as ethical issues,technical difficulties in cell isolation, and the need for long-termimmunosuppressant administration to transplant recipients; thelimitations of using fetal tissue sources have been set forth above.These challenges have hindered the applicability of hNSCs for human use.

Bone marrow (BM) contains stem cells involved not only in hematopoiesisbut also for production of a variety of nonhematopoietic tissues. Asubset of stromal cells in bone marrow, mesenchymal stem cells (MSCs),is capable of self-renewing and producing multiple mesenchymal celllineages, including bone, cartilage, fat tendons, and other connectivetissues (Majumdar et al., 1998, J Cell Physiol. 176:57-66, Pereira etal., 1995, Proc Natl Acad Sci USA. 92: 4857-61, Pittenger et al., 1999,Science 284: 143-7). Bone marrow mesenchymal stem cells normally are notcommitted to the neural lineage in differentiation. Although adult stemcells continue to possess some degrees of multipotency, cell typesproduced from adult stem cells are thought to be limited by theirtissue-specific character. To overcome this barrier, it is necessary toalter the cell lineage of these adult stem cells.

Whilst reducing the present invention to practice, the present inventorshave found that out of a vast number of potential micro RNAs (miRNAs),only particular miRNAs may be regulated in order to induce neural stemcell differentiation of mesenchymal stem cells (MSCs) and propose thatsuch differentiated MSCs may be used to treat patients with braindiseases or disorders.

Further, the present inventors identified particular combinations ofmiRNAs whose regulation was found to synergistically increase thedifferentiation towards NSCs, as measured by nestin and SOX-2expression.

Whilst further reducing the present invention to practice the presentinventors uncovered that upon manipulation of the miRNA expression ofNSCs, cells expressing motor neurons markers may be generated.

Thus, the present inventors showed that up-regulation of at least one ofmiR-368, miR-302b, miR-365-3p, miR-365-5p, miR-Let-7a, miR-Let-7b,miR-218, miR-134, miR-124, miR-125a, miR-9, miR-154, miR-20a, miR-130ain neural stem cells (NSCs), induced a motor neuron phenotype, whilstdown-regulation of at least one of miR-372, miR-373, miR-141, miR-199a,miR-32, miR-33, miR-221 and miR-223 in NSCs also induced a motor neuronphenotype.

Further, the present inventors identified particular combinations ofmiRNAs whose regulation was found to synergistically increase thedifferentiation towards motor neurons, as measured by expression ofmotor neuron markers including islet1, HB9 and the neuronal markersneurofilament and tubulin.

Thus, according to one aspect of the present invention there is provideda method of predisposing mesenchymal stem cells to differentiate intoneural stem cells, the method comprising up-regulating a level of atleast one exogenous miRNA selected from the group consisting of miR302b,miR-371, miR-134, miR-219, miR-154, miR-155, miR-32, miR-33, miR-126,miR-127, miR-132, miR-137, miR-572, miR-935a, miR-891a, miR-1202,miR-1275, let-7c, miR-665, miR-4258, miR-361-3p, miR-374a-star,miR-892b, miR-361-5p, miR-181a, miR-16, miR-636, miR-4284, miR-1208,miR-1274b, miR-30c-2-star, miR-501-3p, hsa-miR-92a, miR-378b, miR-1287,miR-425-star, miR-324-5p, miR-3178, miR-219-1-3p, miR-197, miR-181b,miR-500-star, miR-106b, miR-502-3p, miR-30c, miR-1275, miR-422a, miR-93,miR-181d, miR-1307, miR-1301, miR-99a, miR-505-star, miR-1202, miR-12,miR-532-5p, miR-195, miR-532-3p, miR-106a, miR-17, miR-1271, miR-769-3p,miR-15b, miR-324-3p, miR-20a, miR-501-5p, miR-330-3p, miR-874, miR-500,miR-25, miR-769-5p, miR-125b-2-star, miR-130b, miR-504, miR-181a-2-star,miR-885-3p, miR-1246, miR-92b, miR-362-5p, miR-572, miR-4270, miR-378c,miR-93-star, miR-149, miR-363, miR-9, miR-18a, miR-891a, miR-346,miR-124, miR-497, miR-378, miR-1231, miR-139-5p, miR-3180-3p,miR-9-star, miR-935 and miR-20b in mesenchymal stem cells (MSCs),thereby predisposing mesenchymal stem cells to differentiate into theneural stem cells.

As used herein, the phrase “predisposing MSCs to differentiate intoneural stem cells (NSCs)” refers to causing the MSCs to differentiatealong the NSC lineage. The generated cells may be fully differentiatedinto NSCs, or partially differentiated into NSCs.

The phrase “at least one” as used in the specification refers to one,two, three four, five six, seven, eight, nine, ten or more miRNAs.Examples of particular combinations of miRNAs are provided herein below.

Mesenchymal stem cells give rise to one or more mesenchymal tissues(e.g., adipose, osseous, cartilaginous, elastic and fibrous connectivetissues, myoblasts) as well as to tissues other than those originatingin the embryonic mesoderm (e.g., neural cells) depending upon variousinfluences from bioactive factors such as cytokines. Although such cellscan be isolated from embryonic yolk sac, placenta, umbilical cord, fetaland adolescent skin, blood and other tissues, their abundance in theeasily accessible fat tissue and BM far exceeds their abundance in othertissues and as such isolation from BM and fat tissue is presentlypreferred.

Methods of isolating, purifying and expanding mesenchymal stem cells(MSCs) are known in the arts and include, for example, those disclosedby Caplan and Haynesworth in U.S. Pat. No. 5,486,359 and Jones E. A. etal., 2002, Isolation and characterization of bone marrow multipotentialmesenchymal progenitor cells, Arthritis Rheum. 46(12): 3349-60.

Mesenchymal stem cells may be isolated from various tissues includingbut not limited to bone marrow, peripheral blood, blood, chorionic andamniotic placenta (e.g. fetal side of the placenta), cord blood,umbilical cord, amniotic fluid, placenta and from adipose tissue.

A method of isolating mesenchymal stem cells from peripheral blood isdescribed by Kassis et al [Bone Marrow Transplant. 2006 May;37(10):967-76]. A method of isolating mesenchymal stem cells fromplacental tissue is described by Zhang et al [Chinese Medical Journal,2004, 117 (6):882-887]. Methods of isolating and culturing adiposetissue, placental and cord blood mesenchymal stem cells are described byKern et al [Stem Cells, 2006; 24:1294-1301].

According to a preferred embodiment of this aspect of the presentinvention, the mesenchymal stem cells are human.

According to another embodiment of this aspect of the present invention,the mesenchymal stem cells are isolated from placenta and umbilical cordof newborn humans.

Bone marrow can be isolated from the iliac crest of an individual byaspiration. Low-density BM mononuclear cells (BMMNC) may be separated bya FICOL-PAQUE density gradient or by elimination of red blood cellsusing Hetastarch (hydroxyethyl starch). Preferably, mesenchymal stemcell cultures are generated by diluting BM aspirates (usually 20 ml)with equal volumes of Hank's balanced salt solution (HBSS; GIBCOLaboratories, Grand Island, N.Y., USA) and layering the diluted cellsover about 10 ml of a Ficoll column (Ficoll-Paque; Pharmacia,Piscataway, N.J., USA). Following 30 minutes of centrifugation at2,500×g, the mononuclear cell layer is removed from the interface andsuspended in HBSS. Cells are then centrifuged at 1,500×g for 15 minutesand resuspended in a complete medium (MEM, a medium withoutdeoxyribonucleotides or ribonucleotides; GIBCO); 20% fetal calf serum(FCS) derived from a lot selected for rapid growth of MSCs (AtlantaBiologicals, Norcross, Ga.); 100 units/ml penicillin (GIBCO), 100 μg/mlstreptomycin (GIBCO); and 2 mM L-glutamine (GIBCO). Resuspended cellsare plated in about 25 ml of medium in a 10 cm culture dish (CorningGlass Works, Corning, N.Y.) and incubated at 37° C. with 5% humidifiedC02. Following 24 hours in culture, non-adherent cells are discarded,and the adherent cells are thoroughly washed twice with phosphatebuffered saline (PBS). The medium is replaced with a fresh completemedium every 3 or 4 days for about 14 days. Adherent cells are thenharvested with 0.25% trypsin and 1 mM EDTA (Trypsin/EDTA, GIBCO) for 5min at 37° C., re-plated in a 6-cm plate and cultured for another 14days. Cells are then trypsinized and counted using a cell countingdevice such as for example, a hemocytometer (Hausser Scientific,Horsham, Pa.). Cultured cells are recovered by centrifugation andresuspended with 5% DMSO and 30% FCS at a concentration of 1 to 2×106cells per ml. Aliquots of about 1 ml each are slowly frozen and storedin liquid nitrogen.

Adipose tissue-derived MSCs can be obtained by liposuction andmononuclear cells can be isolated manually by removal of the fat and fatcells, or using the Celution System (Cytori Therapeutics) following thesame procedure as described above for preparation of MSCs.

According to one embodiment the populations are plated on polystyreneplastic surfaces (e.g. in a flask) and mesenchymal stem cells areisolated by removing non-adherent cells. Alternatively, mesenchymal stemcell may be isolated by FACS using mesenchymal stem cell markers.

Preferably the MSCs are at least 50% purified, more preferably at least75% purified and even more preferably at least 90% purified.

To expand the mesenchymal stem cell fraction, frozen cells are thawed at37° C., diluted with a complete medium and recovered by centrifugationto remove the DMSO. Cells are resuspended in a complete medium andplated at a concentration of about 5,000 cells/cm”. Following 24 hoursin culture, non-adherent cells are removed and the adherent cells areharvested using Trypsin/EDT A, dissociated by passage through a narrowedPasteur pipette, and preferably re-plated at a density of about 1.5 toabout 3.0 cells/cm”. Under these conditions, MSC cultures can grow forabout 50 population doublings and be expanded for about 2000 fold[Colter D C., et al. Rapid expansion of recycling stem cells in culturesof plastic-adherent cells from human bone marrow. Proc Natl Acad SciUSA. 97: 3213-3218, 2000].

MSC cultures utilized by some embodiments of the invention preferablyinclude three groups of cells which are defined by their morphologicalfeatures: small and agranular cells (referred to as RS-1, herein below),small and granular cells (referred to as RS-2, herein below) and largeand moderately granular cells (referred to as mature MSCs, hereinbelow). The presence and concentration of such cells in culture can beassayed by identifying a presence or absence of various cell surfacemarkers, by using, for example, immunofluorescence, in situhybridization, and activity assays.

When MSCs are cultured under the culturing conditions of someembodiments of the invention they exhibit negative staining for thehematopoietic stem cell markers CD34, CD11B, CD43 and CD45. A smallfraction of cells (less than 10%) are dimly positive for CD31 and/orCD38 markers. In addition, mature MSCs are dimly positive for thehematopoietic stem cell marker, CD11 7 (c-Kit), moderately positive forthe osteogenic MSCs marker, Stro-1 [Simmons, P. J. & Torok-Storb, B.(1991). Blood 78, and positive for the thymocytes and peripheral Tlymphocytes marker, CD90 (Thy-1). On the other hand, the RS-1 cells arenegative for the CD117 and Strol markers and are dimly positive for theCD90 marker, and the RS-2 cells are negative for all of these markers.

The mesenchymal stem cells of the present invention may be ofautologous, syngeneic or allogeneic related (matched siblings orhaploidentical family members) or unrelated fully mismatched source, asfurther described herein below.

Culturing of the mesenchymal stem cells can be performed in any mediathat supports neural stem cell differentiation (or at least does notprevent neural stem cell differentiation) such as those described inU.S. Pat. No. 6,528,245 and by Sanchez-Ramos et al. (2000); Woodburry etal. (2000); Woodburry et al. (J. Neurisci. Res. 96:908-917, 2001); Blackand Woodbury (Blood Cells Mol. Dis. 27:632-635, 2001); Deng et al.(2001), Kohyama et al. (2001), Reyes and Verfatile (Ann. N.Y. Acad. Sci.30 938:231-235, 2001) and Jiang et al. (Nature 418:47-49, 2002).

The differentiating media may be 05, neurobasal medium, DMEM orDMEM/F12, OptiMEM™ or any other medium that supports neuronal growth.

As mentioned, the mesenchymal stem cells are contacted (either ex vivoor in vivo) with at least one of the following miRNAs in order to inducedifferentiation into neural stem cells-miR302b, miR-371, miR-134,miR-219, miR-154, miR-155, miR-32, miR-33, miR-126, miR-127, miR-132,miR-137, miR-572, miR-935a, miR-891a, miR-1202, miR-1275, let-7c,miR-665, miR-4258, miR-361-3p, miR-374a-star, miR-892b miR-361-5p,miR-181a, miR-16, miR-636, miR-4284, miR-1208, miR-1274b,miR-30c-2-star, miR-501-3p, hsa-miR-92a, miR-378b, miR-1287,miR-425-star, miR-324-5p, miR-3178, miR-219-1-3p, miR-197, miR-181b,miR-500-star, miR-106b, miR-502-3p, miR-30c, miR-1275, miR-422a, miR-93,miR-181d, miR-1307, miR-1301, miR-99a, miR-505-star, miR-1202, miR-12,miR-532-5p, miR-195, miR-532-3p, miR-106a, miR-17, miR-1271, miR-769-3p,miR-15b, miR-324-3p, miR-20a, miR-501-5p, miR-330-3p, miR-874, miR-500,miR-25, miR-769-5p, miR-125b-2-star, miR-130b, miR-504, miR-181a-2-star,miR-885-3p, miR-1246, miR-92b, miR-362-5p, miR-572, miR-4270, miR-378c,miR-93-star, miR-149, miR-363, miR-18a, miR-891a, miR-346, miR-497,miR-378, miR-1231, miR-139-5p, miR-3180-3p, miR-935 and miR-20b.

According to a particular embodiment, the miRNA is selected from thegroup consisting of miR302b, miR-371, miR-134, miR-219, miR-154,miR-155, miR-32, miR-33, miR-126, miR-127, miR-132.

According to another embodiment, the miRNA is selected from the groupconsisting of miR-20b, miR-925, miR-891 and miR-378.

The present invention also contemplates differentiation of mesenchymalstem cells towards a neural stem cell phenotype by down-regulation ofparticular miRNAs—namely miR-1Ob, miR-142-3p, miR-131a, miR-125b,miR-153 and miR-181a.

The present invention contemplates down-regulation of additional miRNAsfor the differentiation of MSCs towards a neural stem cell phenotype.These miRNAs include miR-409-5p, miR-193a-5p, miR-4317, miR-4288,miR-145, miR-143, miR-214, miR-199a-3p, miR-199a-5p, miR-199b-3p,miR-138, miR-31, miR-21, miR-193a-5p, miR-224-star, miR-196a, miR-487b,miR-409-5p, miR-193b-star, miR-379, miR-21-star, miR-27a-star, miR-27a,miR-4317, miR-193b, miR-27b, miR-22, 574-3p, miR-30 4288, miR-23a,miR-221-star, miR-2113, let-7i, miR-24, miR-23b, miR-299-3p,miR-518c-star, miR-221, miR-431-star, miR-523, miR-4313, miR-559,miR-614, miR-653, miR-2278, miR-768-5p, miR-154-star, miR-302a-star,miR-3199 and miR-3137.

According to a particular embodiment, the miRNA which is to bedownregulated is selected from the group consisting of miR-138, miR-214,miR-199a and miR-199b.

Down-regulating such miRNAs can be affected using a polynucleotide whichis hybridizable in cells under physiological conditions to the miRNA.

According to a particular embodiment, the cell population is generatedby up-regulating an expression of miR-124 in mesenchymal stem cells(MSCs) whilst simultaneously down-regulating an expression of miR-let-7in the population of MSCs.

According to a particular embodiment, the cell population is generatedby down-regulating an expression of miR-891 in mesenchymal stem cells(MSCs) whilst simultaneously down-regulating an expression of miR-138 inthe population of MSCs.

According to a particular embodiment, the cell population is generatedby up-regulating an expression of miR-20b in mesenchymal stem cells(MSCs) whilst simultaneously down-regulating an expression of miR-138 inthe population of MSCs.

According to a particular embodiment, the cell population is generatedby up-regulating an expression of miR-378 in mesenchymal stem cells(MSCs) whilst simultaneously down-regulating an expression of miR-138 inthe population of MSCs.

As used herein, the term “hybridizable” refers to capable ofhybridizing, i.e., forming a double strand molecule such as RNA:RNA,RNA:DNA and/or DNA:DNA molecules. “Physiological conditions” refer tothe conditions present in cells, tissue or a whole organism or body.Preferably, the physiological conditions used by the present inventioninclude a temperature between 34-40° C., more preferably, a temperaturebetween 35-38° C., more preferably, a temperature between 36 and 37.5°C., most preferably, a temperature between 37 to 37.5° C.; saltconcentrations (e.g., sodium chloride NaCl) between 0.8-1%, morepreferably, about 0.9%; and/or pH values in the range of 6.5-8, morepreferably, 6.5-7.5, most preferably, pH of 7-7.5.

As mentioned, the present inventors have also uncovered that uponmanipulation of particular miRNAs in neural stem cells, cells expressingmotor neurons markers may be generated.

Thus, according to another aspect of the present invention there isprovided a method of predisposing neural stem cells to differentiateinto motor neurons comprising up-regulating a level of at least oneexogenous miRNA selected from the group consisting of miR-368, miR-302b,miR-365-3p, miR-365-5p, miR-Let-7a, miR-Let-7b, miR-218, miR-134,miR-124, miR-125a, miR-9, miR-154, miR-20a, miR-130a in neural stemcells (NSCs).

The neural stem cells of this aspect of the present invention may benon-committed neural stem cells that are not committed to any particulartype of neural cell such as but not limited to neuronal and glial celltypes. Preferably these cells have a potential to commit to a neuralfate. Alternatively, the neural stem cells may be committed to aparticular neural cell type, such as a motor neuron, but do notexpress/secrete markers of terminal differentiation e.g. do not secreteneurotransmitters.

According to a particular embodiment, the neural stem cells express atleast one of nestin and/or SOX-2. Additional markers include SOX1, SOX3,PSA-NCAM and MUSASHI-1.

Methods of confirming expression of the markers are provided hereinbelow. Formation of “neural rosettes” is another morphologic marker ofneural stem cell formation.

According to one embodiment, the neural stem cells have been generatedby ex vivo differentiation of mesenchymal stem cells or embryonic stemcells (or induced embryonic stem cells).

Mesenchymal stem cells have been described herein above. Numerousmethods are known in the art for differentiating MSCs towards a neuralstem cell fate including genetic modification and/or culturing in amedium which promotes differentiation towards that fate. The mediumtypically comprises growth factors and/or cytokines including, but notlimited to epidermal growth factor (EGF), basic fibroblast growth factor(bFGF). Typically, the differentiation is affected in serum free medium,or serum replacements.

According to a particular embodiment, NSCs are generated by geneticallymodifying the MSCs to express an exogenous miRNA, as described hereinabove.

The phrase “embryonic stem cells” refers to embryonic cells which arecapable of differentiating into cells of all three embryonic germ layers(i.e., endoderm, ectoderm and mesoderm), or remaining in anundifferentiated state. The phrase “embryonic stem cells” may comprisecells which are obtained from the embryonic tissue formed aftergestation (e.g., blastocyst) before implantation of the embryo (i.e., apre-implantation blastocyst), extended blastocyst cells (EBCs) which areobtained from a post-implantation/pre-gastrulation stage blastocyst (seeW02006/040763) and embryonic germ (EG) cells which are obtained from thegenital tissue of a fetus any time during gestation, preferably before10 weeks of gestation.

Induced pluripotent stem cells (iPS; embryonic-like stem cells), arecells obtained by de-differentiation of adult somatic cells which areendowed with pluripotency (i.e., being capable of differentiating intothe three embryonic germ cell layers, i.e., endoderm, ectoderm andmesoderm). According to some embodiments of the invention, such cellsare obtained from a differentiated tissue (e.g., a somatic tissue suchas skin) and undergo de-differentiation by genetic manipulation whichre-program the cell to acquire embryonic stem cells characteristics.According to some embodiments of the invention, the induced pluripotentstem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4and c-Myc in a somatic stem cell.

The embryonic stem cells of some embodiments of the invention can beobtained using well-known cell-culture methods. For example, humanembryonic stem cells can be isolated from human blastocysts. Humanblastocysts are typically obtained from human in vivo pre-implantationembryos or from in vitro fertilized (IVF) embryos. Alternatively, asingle cell human embryo can be expanded to the blastocyst stage. Forthe isolation of human ES cells the zona pellucida is removed from theblastocyst and the inner cell mass (ICM) is isolated by immunosurgery,in which the trophectoderm cells are lysed and removed from the intactICM by gentle pipetting. The ICM is then plated in a tissue cultureflask containing the appropriate medium which enables its outgrowth.Following 9 to 15 days, the ICM derived outgrowth is dissociated intoclumps either by a mechanical dissociation or by an enzymaticdegradation and the cells are then re-plated on a fresh tissue culturemedium. Colonies demonstrating undifferentiated morphology areindividually selected by micropipette, mechanically dissociated intoclumps, and re-plated. Resulting ES cells are then routinely split every4-7 days. For further details on methods of preparation human ES cellssee Thomson et al., [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998;Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92:7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 30 1989]; and Gardner etal., [Fertil. Steril. 69: 84, 1998].

It will be appreciated that commercially available stem cells can alsobe used with this aspect of some embodiments of the invention. Human EScells can be purchased from the NIH human embryonic stem cells registry(www.escr.nih.gov). Non-limiting examples of commercially availableembryonic stem cell lines are BGO1, BG02, BG03, BG04, CY12, CY30, CY92,CY1O, TE03 and TE32.

In addition, ES cells can be obtained from other species as well,including mouse (Mills and Bradley, 2001), golden hamster [Doetschman etal., 1988, Dev Biol. 127: 224-7], rat [Iannaccone et al., 1994, DevBiol. 163: 288-92] rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8;Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33], severaldomestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl.43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova etal., 2001, Cloning. 3: 59-67] and non-human primate species (Rhesusmonkey and marmoset) [Thomson et al., 1995, Proc Natl Acad Sci USA. 92:7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

Induced pluripotent stem cells (iPS) (embryonic-like stem cells) can begenerated from somatic cells by genetic manipulation of somatic cells,e.g., by retroviral transduction of somatic cells such as fibroblasts,hepatocytes, gastric epithelial cells with transcription factors such asOct-3/4, Sox2, c-Myc, and KLF4 [Yamanaka S, Cell Stem Cell. 2007,1(1):39-49; Aoi T, et al., Generation of Pluripotent Stem Cells fromAdult Mouse Liver and Stomach Cells. Science. 2008 February 14. (Epubahead of print); IH Park, Zhao R, West J A, et al. Reprogramming ofhuman somatic cells to pluripotency with defined factors. Nature 2008;451:141-146; K Takahashi, Tanabe K, Ohnuki M, et al. Induction ofpluripotent stem cells from adult human fibroblasts by defined factors.Cell 2007; 131:861-872]. Other embryonic-like stem cells can begenerated by nuclear transfer to oocytes, fusion with embryonic stemcells or nuclear transfer into zygotes if the recipient cells arearrested in mitosis.

Methods of generating neural stem cells from ESCs or iPS cells are knownin the art and include for example those which induce differentiationvia embryoid bodies and those which induce differentiation via adherentculture. Particular protocols for differentiating ESCs towards aneuronal fate are reviewed in Dhara et al., Journal of CellularBiochemistry 105:633-640 (2008), the contents of which are incorporatedherein by reference. It will be appreciated that many other methods areknown for differentiating ESC, iPSCs and MSCs towards neuronal stemcells and the present application contemplates use of all these methods.

The neuronal stem cells of the present invention may be of autologous,syngeneic or allogeneic related (matched siblings or haploidenticalfamily members) or unrelated fully mismatched source.

Culturing of neuronal stem cells can be performed in any media thatsupports neural stem cell differentiation, examples of which aredescribed herein above.

As mentioned, the neuronal stem cells are contacted (either ex vivo orin vivo) with at least one of the following miRNAs in order to inducedifferentiation towards the motor neuron lineage—miR-368, miR-302b,miR-365-3p, miR-365-5p, miR-Let-7a miR-Let-7b, miR-218, miR-134,miR-124, miR-125a, miR-9, miR-154, miR-20a and miR-130a.

The present invention also contemplates differentiation of neuronal stemcells towards motor neuron phenotype by down-regulation of particularmiRNAs—namely miR-372, miR-373, miR-141, miR-199a, miR-32, miR-33,miR-221 and miR-223.

Down-regulating such miRNAs can be affected using a polynucleotide whichis hybridizable in cells under physiological conditions to the miRNAmolecule.

According to a particular embodiment, the cell population is generatedby up-regulating an expression of each of miR Let-7a, miR-124, miR-368and miR-154 in the neural stem cells.

According to a particular embodiment, the cell population is generatedby up-regulating an expression of each of miR-125a, miR-9 and miR-130ain the neural stem cells.

According to still another embodiment, the cell population is generatedby up-regulating an expression of each of each of miR-218, miR-134 andmiR-20a.

The present inventors further contemplate down-regulating each ofmiR-141, miR-32, miR-33, miR-221, miR-223 and miR-373 in addition to anyof the methods described herein above to enhance the differentiationtowards the motor neuron phenotype.

Mesenchymal stem cells were differentiated into motor neurons byoverexpressing Olig2 and HB9. The present inventors performed a miRNAarray analysis on the differentiated and non-differentiated cells andfound a number of miRNAs that were overexpressed in a statisticallysignificant manner (more than 3 fold) and a number of miRNAs that weredownregulated in a statistically significant manner (more than 3 fold).The present inventors contemplate that the miRNAs whose expression wasincreased in the differentiated cells may be candidates foroverexpression in order to generate motor neurons from MSCs. The presentinventors contemplate that the miRNAs whose expression was decreased inthe differentiated cells are candidates for downregulation in order togenerate motor neurons from MSCs.

Thus, according to still another aspect of the present invention thereis provided a method of predisposing MSCs to differentiate into motorneurons, the method comprising up-regulating a level of at least oneexogenous miRNA selected from the group consisting of miR-368, miR-365,miR-500, miR-648, miR-491, miR-218, miR-155, miR-192, let-7b, miR-16,miR-210, miR-197, miR-21, miR-373, miR-27a, miR-122, miR-17, miR-494,miR-449, miR-503, miR-30a, miR-196a, miR-122, miR-7, miR-151-5p, miR-16,miR-22, miR-31, miR-424, miR-1, miR-29c, miR-942, miR-100, miR-520,miR-663a, miR-562, miR-449a, miR-449b-5p, miR-520b, miR-451, miR-532-59,miR-605, miR-504, miR-503, miR-155, miR-34a, miR-16, miR-7b, miR-103,miR-124, miR-1385p, miR-16, miR-330, miR-520, miR-608, miR-708, miR-107,miR-137, miR-132, miR-145, miR-204, miR-125b, miR-224, miR-30a, miR-375,miR-101, miR-106b, miR-128, miR-129-5p, miR-153, miR-203, miR-214,miR-338-3p, miR-346, miR-98, miR-107, miR-141, miR-217, miR-424,miR-449, miR-7, miR-9, miR-93, miR-99a, miR-100, miR-1228, miR-183,miR-185, miR-190, miR-522, miR-650, miR-675, miR-342-3p, miR-31 in themesenchymal stem cells (MSCs).

According to yet another aspect of the present invention there isprovided a method of predisposing MSCs to differentiate into motorneurons, the method comprising down-regulating an expression of at leastone miRNA selected from the group consisting of miR-199a, miR-372,miR-373, miR-942, miR-2113, miR-301a-3p, miR-302c, miR-30b-5p, miR-30c,miR-326, miR-328, miR-331-3p, miR-340, miR-345, miR-361-5p, miR-363,miR-365a-3p, miR-371a-3p, miR-3′73-3p, miR-374a, miR-423-3p,miR-449b-5p, miR-451a, miR-494, miR-504, miR-515-3p, miR-516a-3p,miR-519e, miR-520a-3p, miR-520c-3p, miR-520g, miR-532-5p, miR-559,miR-562, miR-572, miR-590-5p, miR-605, miR-608, miR-626, miR-639,miR-654-3p, miR-657, miR-661, miR-708-5p, miR-942, miR-96, miR-99arnoand miR-194 by up-regulating a level of at least one polynucleotideagent that hybridizes and inhibits a function of said at least one miRNAin the MSCs thereby predisposing MSCs to differentiate into the motorneurons.

The term “microRNA”, “miRNA”, and “miR” are synonymous and refer to acollection of non-coding single-stranded RNA molecules of about 19-28nucleotides in length, which regulate gene expression. miRNAs are foundin a wide range of organisms and have been shown to play a role indevelopment, homeostasis, and disease etiology.

Below is a brief description of the mechanism of miRNA activity.

Genes coding for miRNAs are transcribed leading to production of a miRNAprecursor known as the pri-miRNA. The pri-miRNA is typically part of apolycistronic RNA comprising multiple pri-miRNAs. The pri-miRNA may forma hairpin with a stem and loop. The stem may comprise mismatched bases.

The hairpin structure of the pri-miRNA is recognized by Drosha, which isan RNase III endonuclease. Drosha typically recognizes terminal loops inthe pri-miRNA and cleaves approximately two helical turns into the stemto produce a 60-70 nt precursor known as the pre-miRNA. Drosha cleavesthe pri-miRNA with a staggered cut typical of RNase III endonucleasesyielding a pre-miRNA stem loop with a 5′ phosphate and −2 nucleotide 3′overhang. It is estimated that approximately one helical turn of stem(−10 nucleotides) extending beyond the Drosha cleavage site is essentialfor efficient processing. The pre-miRNA is then actively transportedfrom the nucleus to the cytoplasm by Ran-OTP and the export receptorexportin-5.

The double-stranded stem of the pre-miRNA is then recognized by Dicer,which is also an RNase III endonuclease. Dicer may also recognize the 5′phosphate and 3′ overhang at the base of the stem loop. Dicer thencleaves off the terminal loop two helical turns away from the base ofthe stem loop leaving an additional 5′ phosphate and −2 nucleotide 3′overhang. The resulting siRNA-like duplex, which may comprisemismatches, comprises the mature miRNA and a similar-sized fragmentknown as the miRNA*. The miRNA and miRNA* may be derived from opposingarms of the pri-miRNA and pre-miRNA. miRNA* sequences may be found inlibraries of cloned miRNAs but typically at lower frequency than themiRNAs.

Although initially present as a double-stranded species with miRNA*, themiRNA eventually become incorporated as a single-stranded RNA into aribonucleoprotein complex known as the RNA-induced silencing complex(RISC). Various proteins can form the RISC, which can lead tovariability in specificity for miRNA/miRNA* duplexes, binding site ofthe target gene, activity of miRNA (repress or activate), and whichstrand of the miRN A/miRNA* duplex is loaded in to the RISC.

When the miRNA strand of the miRNA:miRNA* duplex is loaded into theRISC, the miRNA* is removed and degraded. The strand of the miRNA:miRNA*duplex that is loaded into the RISC is the strand whose 5′ end is lesstightly paired. In cases where both ends of the miRNA:miRNA* haveroughly equivalent 5′ pairing, both miRNA and miRNA* may have genesilencing activity.

The RISC identifies target nucleic acids based on high levels ofcomplementarity between the miRNA and the mRNA, especially bynucleotides 2-7 of the miRNA.

A number of studies have looked at the base-pairing requirement betweenmiRNA and its mRNA target for achieving efficient inhibition oftranslation (reviewed by Bartel 2004, Cell 116-281). In mammalian cells,the first 8 nucleotides of the miRNA may be important (Doench & Sharp2004 GenesDev 2004-504). However, other parts of the microRNA may alsoparticipate in mRNA binding. Moreover, sufficient base pairing at the 3′can compensate for insufficient pairing at the 5′ (Brennecke et al, 2005PLoS 3-e85). Computation studies, analyzing miRNA binding on wholegenomes have suggested a specific role for bases 2-7 at the 5′ of themiRNA in target binding but the role of the first nucleotide, foundusually to be “A” was also recognized (Lewis et at 2005 Cell 120-15).Similarly, nucleotides 1-7 or 2-8 were used to identify and validatetargets by Krek et al (2005, Nat Genet 37-495).

The target sites in the mRNA may be in the 5′ UTR, the 3′ UTR or in thecoding region. Interestingly, multiple miRNAs may regulate the same mRNAtarget by recognizing the same or multiple sites. The presence ofmultiple miRNA binding sites in most genetically identified targets mayindicate that the cooperative action of multiple RISCs provides the mostefficient translational inhibition.

miRNAs may direct the RISC to down-regulate gene expression by either oftwo mechanisms: mRNA cleavage or translational repression. The miRNA mayspecify cleavage of the mRNA if the mRNA has a certain degree ofcomplementarity to the miRNA. When a miRNA guides cleavage, the cut istypically between the nucleotides pairing to residues 10 and 11 of themiRNA. Alternatively, the miRNA may repress translation if the miRNAdoes not have the requisite degree of complementarity to the miRNA.Translational repression may be more prevalent in animals since animalsmay have a lower degree of complementarity between the miRNA and bindingsite.

It should be noted that there may be variability in the 5′ and 3′ endsof any pair of miRNA and miRNA*. This variability may be due tovariability in the enzymatic processing of Drosha and Dicer with respectto the site of cleavage. Variability at the 5′ and 3′ ends of miRNA andmiRNA* may also be due to mismatches in the stem structures of thepri-miRNA and pre-miRNA. The mismatches of the stem strands may lead toa population of different hairpin structures. Variability in the stemstructures may also lead to variability in the products of cleavage byDrosha and Dicer.

The term “microRNA mimic” refers to synthetic non-coding RNAs that arecapable of entering the RNAi pathway and regulating gene expression.miRNA mimics imitate the function of endogenous microRNAs (miRNAs) andcan be designed as mature, double stranded molecules or mimic precursors(e.g., or pre-miRNAs). miRNA mimics can be comprised of modified orunmodified RNA, DNA, RNA-DNA hybrids, or alternative nucleic acidchemistries (e.g., LNAs or 2′-0,4′-C-ethylene-bridged nucleic acids(ENA)). Other modifications are described herein below. For mature,double stranded miRNA mimics, the length of the duplex region can varybetween 13-33, 18-20 24 or 21-23 nucleotides. The miRNA may alsocomprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39 or 40 nucleotides. The sequence of the miRNA may bethe first 13-33 nucleotides of the pre-miRNA. The sequence of the miRNAmay also be the last 13-33 nucleotides of the pre-miRNA. The sequence ofthe miRNA may comprise any of the sequences described herein, orvariants thereof.

It will be appreciated from the description provided herein above, thatcontacting mesenchymal stem cells may be affected in a number of ways:

1. Transiently transfecting the mesenchymal stem cells with the maturemiRNA (or modified form thereof, as described herein below). The miRNAsdesigned according to the teachings of the present invention can begenerated according to any oligonucleotide synthesis method known in theart, including both enzymatic syntheses and solid-phase syntheses.Equipment and reagents for executing solid-phase synthesis arecommercially available from, for example, Applied Biosystems. Any othermeans for such synthesis may also be employed; the actual synthesis ofthe oligonucleotides is well within the capabilities of one skilled inthe art and can be accomplished via established methodologies asdetailed in, for example: Sambrook, J. and Russell, D. W. 5 (2001),“Molecular Cloning: A Laboratory Manual”; Ausubel, R. M. et al., eds.(1994, 1989), “Current Protocols in Molecular Biology,” Volumes I-III,John Wiley & Sons, Baltimore, Md.; Perbal, B. (1988), “A Practical Guideto Molecular Cloning,” John Wiley & Sons, New York; and Gait, M. J., ed.(1984), “Oligonucleotide Synthesis”; utilizing solid-phase chemistry,e.g. cyanoethyl phosphoramidite followed by deprotection, desalting, andpurification by, for example, an automated trityl-on method or HPLC.

2. Stably, or transiently transfecting the mesenchymal stem cells withan expression vector which encodes the mature miRNA or with miRNA mimic.

3. Stably, or transiently transfecting the mesenchymal stem cells withan expression vector which encodes the pre-miRNA. The pre-miRNA sequencemay comprise from 45-90, 60-80 or 60-70 nucleotides. The sequence of thepre-miRNA may comprise a miRNA and a miRNA* as set forth herein. Thesequence of the pre-miRNA may also be that of a pri-miRNA excluding from0-160 nucleotides from the 5′ and 3′ ends of the pri-miRNA. The sequenceof the pre-miRNA may comprise the sequence of the miRNA, or variantsthereof.

4. Stably, or transiently transfecting the mesenchymal stem cells withan expression vector which encodes the pri-miRNA. The pri-miRNA sequencemay comprise from 45-30,000, 50-25,000, 100-20,000, 1,000-1,500 or80-100 nucleotides. The sequence of the pri-miRNA may comprise apre-miRNA, miRNA and miRNA*, as set forth herein, and variants thereof.Preparation of miRNAs mimics can be affected by chemical synthesismethods or by recombinant methods.

miRNA antagonists may be introduced into cells using transfectionprotocols known in the art using either siRNAs or expression vectorssuch as Anatgomirs.

As mentioned herein above, the polynucleotides which down-regulate themiRNAs described herein above may be provided as modifiedpolynucleotides using various methods known in the art.

For example, the oligonucleotides (e.g. miRNAs) or polynucleotides ofthe present invention may comprise heterocylic nucleosides consisting ofpurines and the pyrimidines bases, bonded in a 3′-to-5′ phosphodiesterlinkage.

Preferably used oligonucleotides or polynucleotides are those modifiedeither in backbone, internucleoside linkages, or bases, as is broadlydescribed herein under.

Specific examples of preferred oligonucleotides or polynucleotidesuseful according to this aspect of the present invention includeoligonucleotides or polynucleotides containing modified backbones ornon-natural internucleoside linkages. Oligonucleotides orpolynucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide or polynucleotide backbones include,for example: phosphorothioates; chiral phosphorothioates;phosphorodithioates; phosphotriesters; aminoalkyl phosphotriesters;methyl and other alkyl phosphonates, including 3′-alkylene phosphonatesand chiral phosphonates; phosphinates; phosphoramidates, including3′-amino phosphoramidate and aminoalkylphosphoramidates;thionophosphoramidates; thionoalkylphosphonates;thionoalkylphosphotriesters; and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogues of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts, and free acidforms of the above modifications can also be used.

Alternatively, modified oligonucleotide or polynucleotide backbones thatdo not include a phosphorus atom therein have backbones that are formedby short-chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short-chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide,and sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; alkene-containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH2 component parts, as disclosed inU.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677,5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;5,663,312; 5,633,360; 5,677,437; and 5,677,439.

Other oligonucleotides or polynucleotides which may be used according tothe present invention are those modified in both sugar and theintemucleoside linkage, i.e., the backbone of the nucleotide units isreplaced with novel groups. The base units are maintained forcomplementation with the appropriate polynucleotide target. An exampleof such an oligonucleotide mimetic includes a peptide nucleic acid(PNA). A PNA oligonucleotide refers to an oligonucleotide where thesugar-backbone is replaced with an amide-containing backbone, inparticular an aminoethylglycine backbone. The bases are retained and arebound directly or indirectly to aza-nitrogen atoms of the amide portionof the backbone. United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262; each of which is herein incorporated byreference. Other backbone modifications which may be used in the presentinvention are disclosed in U.S. Pat. No. 6,303,374.

Oligonucleotides or polynucleotides of the present invention may alsoinclude base modifications or substitutions. As used herein,“unmodified” or “natural” bases include the purine bases adenine (A) andguanine (G) and the pyrimidine bases thymine (T), cytosine (C), anduracil (U). “Modified” bases include but are not limited to othersynthetic and natural bases, such as: 5-methylcytosine (5-me-C);5-hydroxymethyl cytosine; xanthine; hypoxanthine; 2-aminoadenine;6-methyl and other alkyl derivatives of adenine and guanine; 2-propyland other alkyl derivatives of adenine and guanine; 2-thiouracil,2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azo uracil, cytosine, and thymine; 5-uracil(pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl, and other 8-substituted adenines and guanines; 5-halo,particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracilsand cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and8-azaadenine; 7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and3-deazaadenine. Additional modified bases include those disclosed in:U.S. Pat. No. 3,687,808; Kroschwitz, J. I., ed. (1990),“The ConciseEncyclopedia Of Polymer Science And Engineering,” pages 858-859, JohnWiley & Sons; Englisch et al. (1991), “Angewandte Chemie,” InternationalEdition, 30, 613; and Sanghvi, Y. S., “Antisense Research andApplications,” Chapter 15, pages 289-302, S. T. Crooke and B. Lebleu,eds., CRC Press, 1993. Such modified bases are particularly useful forincreasing the binding affinity of the oligomeric compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines,and N-2, N-6, and 0-6-substituted purines, including2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S. et al. (1993),“Antisense Research and Applications,” pages 276-278, CRC Press, BocaRaton), and are presently preferred base substitutions, even moreparticularly when combined with 2′-0-methoxyethyl sugar modifications.

To express miRNAs or polynucleotide agents which regulate miRNAs inmesencyhymal stem cells or neural stem cells, a polynucleotide sequenceencoding the miRNA (or pre-miRNA, or pri-miRNA, or polynucleotide whichdown-regulates the miRNAs) is preferably ligated into a nucleic acidconstruct suitable for mesenchymal stem cell (or neural stem cell)expression. Such a nucleic acid construct includes a promoter sequencefor directing transcription of the polynucleotide sequence in the cellin a constitutive or inducible manner.

It will be appreciated that the nucleic acid construct of someembodiments of the invention can also utilize miRNA homologues whichexhibit the desired activity (e.g. motor neuron or neural stem celldifferentiating ability). Such homologues can be, for example, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%identical to any of the sequences described herein above, as determinedusing the BestFit software of the Wisconsin sequence analysis package,utilizing the Smith and Waterman algorithm, where gap weight equals 50,length weight equals 3, average match equals 10 and average mismatchequals −9.

In addition, the homologues can be, for example, at least 60%, at least61%, at least 62%, at least 63%, at least 64%, at least 65%, at least66%, at least 67%, at least 68%, at least 69%, at least 70%, at least71%, at least 72%, at least 73%, at least 74%, at least 75%, at least76%, at least 77%, at least 78%, at least 79%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% identical to thesequences described herein above, as determined using the BestFitsoftware of the Wisconsin sequence analysis package, utilizing the Smithand Waterman algorithm, where gap weight equals 50, length weight equals3, average match equals 10 and average mismatch equals −9.

Constitutive promoters suitable for use with some embodiments of theinvention are promoter sequences which are active under mostenvironmental conditions and most types of cells such as thecytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoterssuitable for use with some embodiments of the invention include forexample tetracycline-inducible promoter (Zabala M, et al., Cancer Res.2004, 64(8): 2799-804).

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of someembodiments of the invention is active in the specific cell populationtransformed—i.e. mesenchymal stem cells or neural stem cells.

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for some embodiments of the inventioninclude those derived from polyoma virus, human or murinecytomegalovirus (CMV), the long term repeat from various retrovirusessuch as murine leukemia virus, murine or Rous sarcoma virus and HIV.See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the elements already described, the expression vector ofsome embodiments of the invention may typically contain otherspecialized elements intended to increase the level of expression ofcloned nucleic acids or to facilitate the identification of cells thatcarry the recombinant DNA. For example, a number of animal virusescontain DNA sequences that promote the extra chromosomal replication ofthe viral genome in permissive cell types. Plasmids bearing these viralreplicons are replicated episomally as long as the appropriate factorsare provided by genes either carried on the plasmid or with the genomeof the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

Examples for mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives. Other expression vectors are available from SBI orSigma.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp205. Other exemplary vectors include pMSG, pAV009/A+, pMTO1O/A+,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

As described above, viruses are very specialized infectious agents thathave evolved, in many cases, to elude host defense mechanisms.Typically, viruses infect and propagate in specific cell types. Thetargeting specificity of viral vectors utilizes its natural specificityto specifically target predetermined cell types and thereby introduce arecombinant gene into the infected cell. Thus, the type of vector usedby some embodiments of the invention will depend on the cell typetransformed. The ability to select suitable vectors according to thecell type transformed is well within the capabilities of the ordinaryskilled artisan and as such no general description of selectionconsideration is provided herein. For example, bone marrow cells can betargeted using the human T cell leukemia virus type I (HTLV-1) andkidney cells may be targeted using the heterologous promoter present inthe baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) asdescribed in Liang C Y et al., 2004 (Arch Virol. 149: 51-60).

According to one embodiment, a lentiviral vector is used to transfectthe mesenchymal stem cells or neural stem cells.

Various methods can be used to introduce the expression vector of someembodiments of the invention into mesenchymal stem cells. Such methodsare generally described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Harbor Laboratory, New York (1989,1992), in Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic GeneTherapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., GeneTargeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey ofMolecular Cloning Vectors and Their Uses, Butterworths, Boston Mass.(1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] andinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. Inaddition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 forpositive-negative selection methods.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

Other vectors can be used that are non-viral, such as cationic lipids,polylysine, and dendrimers.

The miRNAs, miRNA mimics and pre-miRs can be transfected into cells alsousing nanoparticles such as gold nanoparticles and by ferric oxidemagnetic NP—see for example Ghosh et al., Biomaterials. 2013 January;34(3):807-16; Crew E, et al., Anal Chem. 2012 Jan. 3; 84(1):26-9.

Other modes of transfection that do not involved integration include theuse of minicircle DNA vectors or the use of PiggyBac transposon thatallows the transfection of genes that can be later removed from thegenome.

As mentioned hereinabove, a variety of prokaryotic or eukaryotic cellscan be used as host-expression systems to express the miRNAs orpolynucleotide agent capable of down-regulating the miRNA of someembodiments of the invention. These include, but are not limited to,microorganisms, such as bacteria transformed with a recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorcontaining the coding sequence; yeast transformed with recombinant yeastexpression vectors containing the coding sequence; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors, such as Ti plasmid, containingthe coding sequence. Mammalian expression systems can also be used toexpress the miRNAs of some embodiments of the invention.

Examples of bacterial constructs include the pET series of E. coliexpression vectors [Studier et al. (1990) Methods in Enzymol.185:60-89).

In yeast, a number of vectors containing constitutive or induciblepromoters can be used, as disclosed in U.S. Pat. No. 5,932,447.Alternatively, vectors can be used which promote integration of foreignDNA sequences into the yeast chromosome.

The conditions used for contacting the mesenchymal stem cells or neuralstem cells are selected for a time period/concentration ofcells/concentration of miRNA/ratio between cells and miRNA which enablethe miRNA (or inhibitors thereof) to induce differentiation thereof. Thepresent invention further contemplates incubation of the stem cells witha differentiation factor which promotes differentiation towards a motorneuron or neural stem cell lineage. The incubation with suchdifferentiation factors may be affected prior to, concomitant with orfollowing the contacting with the miRNA. Examples of such agents areprovided in the Examples section herein below.

Alternatively, or additionally, the mesenchymal stem cells may begenetically modified so as to express such differentiation factors,using expression constructs such as those described herein above.Further, the mesenchymal stem cell can be genetically modified using theCRISPR/Cas9 system, or an equivalent system, to no long express a targetwhich is being silenced/down-regulated, such as for example RTVP-1.

During or following the differentiation step the stem cells may bemonitored for their differentiation state. Cell differentiation can bedetermined upon examination of cell or tissue-specific markers which areknown to be indicative of differentiation.

For example, the neural stem cells may express at least one of nestinand SOX-2. Additional markers include SOX1, SOX3, PSA-NCAM andMUSASHI-1.

Below is a list of markers that may be used to confirm differentiationinto motor neurons: ChAT (choline acetyltransferase), Chox1O, Enl,Even-skipped (Eve) transcription factor, Evx1/2, Fibroblast growthfactor-1 (FGF1 or acidic FGF), HB9, Isl1 (lslet-1), Isl2, Islet1/2,Lim3, p75(NTR) (p75 neurotrophin receptor), REG2, Sim1, SMI32 (SMI-32)and Zfh1.

Tissue/cell specific markers can be detected using immunologicaltechniques well known in the art [Thomson J A et al., (1998). Science282: 1145-7]. Examples include, but are not limited to, flow cytometryfor membrane-bound markers, immunohistochemistry for extracellular andintracellular markers and enzymatic immunoassay, for secreted molecularmarkers.

It will be appreciated that the cells obtained according to the methodsdescribed herein may be enriched for a particular cell type—e.g.progenitor cell type or mature cell type. Thus for example, the time ofdifferentiation may be selected to obtain an earlier progenitor type(e.g. one that expresses at least one of the following markers nestin,olig2 and Sox2) or a later mature cell type (e.g. one that expresses atleast one of the following markers ChAT, islet1, HB9 and J33 tubulin).

Further enrichment of a particular cell type may be affected using cellsorting techniques such as FACS and magnetic sorting.

In addition, cell differentiation can be also followed by specificreporters that are tagged with GFP or RFP and exhibit increasedfluorescence upon differentiation.

By determining the targets of the miRNAs of the present invention thatare proposed for up-regulation, it will be appreciated that the scope ofthe present invention may be broadened to include down-regulation of thetargets by means other than contacting with miRNA. Correspondingly, bydetermining the targets of the miRNAs of the present invention that areproposed for down-regulation, it will be appreciated that the scope ofthe present invention may be broadened to include up-regulation of thetargets.

For example, the present inventors have shown that one of the targets ofmiR-137 is Related to testis-specific, vespid and pathogenesis protein 1(RTVP-1) Thus the present invention contemplates that differentiationtowards the neural stem cell lineage may be affected by down-regulationof this protein.

Thus, according to another aspect of the invention, there is provided amethod of generating neural stem cells, the method comprising contactingmesenchymal stem cells (MSCs) with an agent that down-regulates anamount and/or activity of Related to testis-specific, vespid andpathogenesis protein 1 (RTVP-1), thereby generating the neural stemcells.

Related to testis-specific, vespid and pathogenesis protein 1 (RTVP-1)was cloned from human GBM cell lines by two groups and was termed gliomapathogenesis-related protein-GLIPR1 or RTVP-1 [Rich T, et al., Gene1996; 180: 125-30], incorporated herein by reference. RTVP-1 contains aputative signal peptide, a trans membrane domain and a SCP domain, witha yet unknown function which is also found in other RTVP-1 homologsincluding TPX-1, the venom allergen antigen 5 and group 1 of the plantpathogenesis-related proteins (PR-1).

Down-regulation of RTVP-1 (or any of the other miRNA targets of thepresent invention) can be obtained at the genomic and/or the transcriptlevel using a variety of molecules which interfere with transcriptionand/or translation (e.g., RNA silencing agents, Ribozyme, DNAzyme andantisense), or on the protein level using e.g., antagonists, enzymesthat cleave the polypeptide and the like.

Following is a list of agents capable of down-regulating expressionlevel and/or activity of RTVP-1.

One example of an agent capable of down-regulating RTVP-1 is an antibodyor antibody fragment capable of specifically binding thereto.Preferably, the antibody is capable of being internalized by the celland entering the nucleus.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, and Fvthat are capable of binding to macrophages. These functional antibodyfragments are defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule, can beproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule that can be obtained by treating wholeantibody with pepsin, followed by reduction, to yield an intact lightchain and a portion of the heavy chain; two Fab′ fragments are obtainedper antibody molecule; (3) (Fab′)2, the fragment of the antibody thatcan be obtained by treating whole antibody with the enzyme pepsinwithout subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragmentsheld together by two disulfide bonds; (4) Fv, defined as a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains; and(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Down-regulation of RTVP-1 can be also achieved by RNA silencing. As usedherein, the phrase “RNA silencing” refers to a group of regulatorymechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing(TGS), post-transcriptional gene silencing (PTGS), quelling,co-suppression, and translational repression] mediated by RNA moleculeswhich result in the inhibition or “silencing” of the expression of acorresponding protein-coding gene. RNA silencing has been observed inmany types of organisms, including plants, animals, and fungi.

As used herein, the term “RNA silencing agent” refers to an RNA which iscapable of inhibiting or “silencing” the expression of a target gene. Incertain embodiments, the RNA silencing agent is capable of preventingcomplete processing (e.g., the full translation and/or expression) of anmRNA molecule through a post-transcriptional silencing mechanism. RNAsilencing agents include non-coding RNA molecules, for example RNAduplexes comprising paired strands, as well as precursor RNAs from whichsuch small non-coding RNAs can be generated. In one embodiment, the RNAsilencing agent is capable of inducing RNA interference. In anotherembodiment, the RNA silencing agent is capable of mediatingtranslational repression.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs). The corresponding process in plants iscommonly referred to as post-transcriptional gene silencing or RNAsilencing and is also referred to as quelling in fungi. The process ofpost-transcriptional gene silencing is thought to be anevolutionarily-conserved cellular defense mechanism used to prevent theexpression of foreign genes and is commonly shared by diverse flora andphyla. Such protection from foreign gene expression may have evolved inresponse to the production of double-stranded RNAs (dsRNAs) derived fromviral infection or from the random integration of transposon elementsinto a host genome via a cellular response that specifically destroyshomologous single-stranded RNA or viral genomic RNA.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes. The RNAi response also features anendonuclease complex, commonly referred to as an RNA-induced silencingcomplex (RISC), which mediates cleavage of single-stranded RNA havingsequence complementary to the antisense strand of the siRNA duplex.Cleavage of the target RNA takes place in the middle of the regioncomplementary to the antisense strand of the siRNA duplex.

Accordingly, the present invention contemplates use of dsRNA todown-regulate protein expression from the mRNA.

According to one embodiment, the dsRNA is greater than 30 bp. The use oflong dsRNAs (i.e. dsRNA greater than 30 bp) has been very limited owingto the belief that these longer regions of double stranded RNA willresult in the induction of the interferon and PKR response. However, theuse of long dsRNAs can provide numerous advantages in that the cell canselect the optimal silencing sequence alleviating the need to testnumerous siRNAs; long dsRNAs will allow for silencing libraries to haveless complexity than would be necessary for siRNAs; and, perhaps mostimportantly, long dsRNA could prevent viral escape mutations when usedas therapeutics.

Various studies demonstrate that long dsRNAs can be used to silence geneexpression without inducing the stress response or causing significantoff-target effects—see for example [Strat et al., Nucleic AcidsResearch, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res.Protoc. 2004; 13:115-125; Diallo M., et al., Oligonucleotides. 2003;13:381-392; Paddison P. J., et al., Proc. Natl Acad. Sci. USA. 2002;99:1443-1448; Tran N., et al., FEBS Lett. 2004; 573:127-134].

In particular, the present invention also contemplates introduction oflong dsRNA (over 30 base transcripts) for gene silencing in cells wherethe interferon pathway is not activated (e.g. embryonic cells andoocytes) see for example Billy et al., PNAS 2001, Vol 98, pages14428-14433. and Diallo et al, Oligonucleotides, Oct. 1, 2003, 13(5):381-392. doi: 10.1089/154545703322617069.

The present invention also contemplates introduction of long dsRNAspecifically designed not to induce the interferon and PKR pathways fordown-regulating gene expression. For example, Shinagwa and Ishii [Genes& Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP,to express long double-strand RNA from an RNA polymerase II (Pol II)promoter. Because the transcripts from pDECAP lack both the 5′-capstructure and the 3′-poly(A) tail that facilitate dsRNA export to thecytoplasm, long ds-RNA from pDECAP does not induce the interferonresponse.

Another method of evading the interferon and PKR pathways in mammaliansystems is by introduction of small inhibitory RNAs (siRNAs) either viatransfection or endogenous expression.

The term “siRNA” refers to small inhibitory RNA duplexes (generallybetween 18-30 basepairs) that induce the RNA interference (RNAi)pathway. Typically, siRNAs are chemically synthesized as 21 mers with acentral 19 bp duplex region and symmetric 2-base 3′-overhangs on thetermini, although it has been recently described that chemicallysynthesized RNA duplexes of 25-30 base length can have as much as a100-fold increase in potency compared with 21 mers at the same location.The observed increased potency obtained using longer RNAs in triggeringRNAi is theorized to result from providing Dicer with a substrate (27mer) instead of a product (21 mer) and that this improves the rate orefficiency of entry of the siRNA duplex into RISC.

It has been found that position of the 3′-overhang influences potency ofan siRNA and asymmetric duplexes having a 3′-overhang on the antisensestrand are generally more potent than those with the 3′-overhang on thesense strand (Rose et al., 2005). This can be attributed to asymmetricalstrand loading into RISC, as the opposite efficacy patterns are observedwhen targeting the antisense transcript.

The strands of a double-stranded interfering RNA (e.g., an siRNA) may beconnected to form a hairpin or stem-loop structure (e.g., an shRNA).Thus, as mentioned the RNA silencing agent of the present invention mayalso be a short hairpin RNA (shRNA).

The term “shRNA”, as used herein, refers to an RNA agent having astem-loop structure, comprising a first and second region ofcomplementary sequence, the degree of complementarity and orientation ofthe regions being sufficient such that base pairing occurs between theregions, the first and second regions being joined by a loop region, theloop resulting from a lack of base pairing between nucleotides (ornucleotide analogs) within the loop region. The number of nucleotides inthe loop is a number between and including 3 to 23, or 5 to 15, or 7 to13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can beinvolved in base-pair interactions with other nucleotides in the loop.Examples of oligonucleotide sequences that can be used to form the loopinclude 5′-UUCAAGAGA-3; (Brummelkamp, T. R. et al. (2002) Science 296:550) and 5′-UUUGUGUAG-3′ (Castanotto, D. et al. (2002) RNA 8:1454). Itwill be recognized by one of skill in the art that the resulting singlechain oligonucleotide forms a stem-loop or hairpin structure comprisinga double-stranded region capable of interacting with the RNAi machinery.

According to another embodiment the RNA silencing agent may be a miRNA,as further described herein above.

Synthesis of RNA silencing agents suitable for use with the presentinvention can be affected as follows. First, the miRNA target mRNAsequence (e.g. CTGF sequence) is scanned downstream of the AUG startcodon for AA dinucleotide sequences. Occurrence of each AA and the 3′adjacent 19 nucleotides is recorded as potential siRNA target sites.Preferably, siRNA target sites are selected from the open reading frame,as untranslated regions (UTRs) are richer in regulatory protein bindingsites. UTR-binding proteins and/or translation initiation complexes mayinterfere with binding of the siRNA endonuclease complex [TuschlChemBiochem. 2:239-245]. It will be appreciated though, that siRNAsdirected at untranslated regions may also be effective, as demonstratedfor GAPDH wherein siRNA directed at the 5′ UTR mediated about 90%decrease in cellular GAPDH mRNA and completely abolished protein level(www.ambion.com/techlib/tn/91/912.html).

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server(www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibitsignificant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene forevaluation. For better evaluation of the selected siRNAs, a negativecontrol is preferably used in conjunction. Negative control siRNApreferably include the same nucleotide composition as the siRNAs butlack significant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

The RNA silencing agents of the present invention may comprise nucleicacid analogs that may have at least one different linkage, e.g.,phosphoramidate, phosphorothioate, phosphorodithioate, or0-methylphosphoroamidite linkages and peptide nucleic acid backbones andlinkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which areincorporated by reference. Nucleic acids containing one or morenon-naturally occurring or modified nucleotides are also included withinone definition of nucleic acids. The modified nucleotide analog may belocated for example at the 5′-end and/or the 3′-end of the nucleic acidmolecule. Representative examples of nucleotide analogs may be selectedfrom sugar- or backbone-modified ribonucleotides. It should be noted,however, that also nucleobase-modified ribonucleotides, i.e.ribonucleotides, containing a non-naturally occurring nucleobase insteadof a naturally occurring nucleobase such as uridines or cytidinesmodified at the 5-position, e.g. 5-(2-amino) propyl uridine, 5-bromouridine; adenosines and guanosines modified at the 8-position, e.g.8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; 0- andN-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The2′-0H-group may be replaced by a group selected from H, OR, R, halo, SH,SR, NH2, NHR, NR2 or CN, wherein R is C1-C6 alkyl, alkenyl or alkynyland halo is F, Cl, Br or I. Modified nucleotides also includenucleotides conjugated with cholesterol through, e.g., a hydroxyprolinollinkage as described in Krutzfeldt et al., Nature 438:685-689 (2005),Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent PublicationNo. 20050107325, which are incorporated herein by reference. Additionalmodified nucleotides and nucleic acids are described in U.S. PatentPublication No. 20050182005, which is incorporated herein by reference.Modifications of the ribose-phosphate backbone may be done for a varietyof reasons, e.g., to increase the stability and half-life of suchmolecules in physiological environments, to enhance diffusion acrosscell membranes, or as probes on a biochip. The backbone modification mayalso enhance resistance to degradation, such as in the harsh endocyticenvironment of cells. The backbone modification may also reduce nucleicacid clearance by hepatocytes, such as in the liver and kidney. Mixturesof naturally occurring nucleic acids and analogs may be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

In some embodiments, the RNA silencing agent provided herein can befunctionally associated with a cell-penetrating peptide.” As usedherein, a “cell-penetrating peptide” is a peptide that comprises a short(about 12-30 residues) amino acid sequence or functional motif thatconfers the energy-independent (i.e., non-endocytotic) translocationproperties associated with transport of the membrane-permeable complexacross the plasma and/or nuclear membranes of a cell. Thecell-penetrating peptide used in the membrane-permeable complex of thepresent invention preferably comprises at least one non-functionalcysteine residue, which is either free or derivatized to form adisulfide link with a double-stranded ribonucleic acid that has beenmodified for such linkage. Representative amino acid motifs conferringsuch properties are listed in U.S. Pat. No. 6,348,185, the contents ofwhich are expressly incorporated herein by reference. Thecell-penetrating peptides of the present invention preferably include,but are not limited to, penetratin, transportan, plsl, TAT(48-60), pVEC,MTS, and MAP.

Another agent capable of down-regulating RTVP-1 is a DNAzyme moleculecapable of specifically cleaving an mRNA transcript or DNA sequence ofCTGF. DNAzymes are single-stranded polynucleotides which are capable ofcleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995; 2:655; Santoro, S. W. &Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262) A general model(the “10-23” model) for the DNAzyme has been proposed. “10-23” DNAzymeshave a catalytic domain of 15 deoxyribonucleotides, flanked by twosubstrate-recognition domains of seven to nine deoxyribonucleotideseach. This type of DNAzyme can effectively cleave its substrate RNA atpurine:pyrimidine junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl,Acad. Sci. USA 20 199; for rev of DNAzymes see Khachigian, L M [CurrOpin Mol Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al.

Down-regulation of RTVP-1 can also be obtained by using an antisensepolynucleotide capable of specifically hybridizing with an mRNAtranscript encoding RTVP-1.

Design of antisense molecules which can be used to efficientlydown-regulate RTVP-1 should take into consideration two aspectsimportant to the antisense approach. The first aspect is delivery of theoligonucleotide into the cytoplasm of the appropriate cells, while thesecond aspect is design of an oligonucleotide which specifically bindsthe designated mRNA within cells in a way which inhibits translationthereof.

The prior art teaches of a number of delivery strategies which can beused to efficiently deliver oligonucleotides into a wide variety of celltypes [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett etal. Blood 91: 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40(1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) andAoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997)].

In addition, algorithms for identifying those sequences with the highestpredicted binding affinity for their target mRNA based on athermodynamic cycle that accounts for the energetics of structuralalterations in both the target mRNA and the oligonucleotide are alsoavailable [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9(1999)].

Such algorithms have been successfully used to implement an antisenseapproach in cells. For example, the algorithm developed by Walton et al.enabled scientists to successfully design antisense oligonucleotides forrabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNFalpha) transcripts. The same research group has more recently reportedthat the antisense activity of rationally selected oligonucleotidesagainst three model target mRNAs (human lactate dehydrogenase A and Band rat gp 130) in cell culture as evaluated by a kinetic PCR techniqueproved effective in almost all cases, including tests against threedifferent targets in two cell types with phosphodiester andphosphorothioate oligonucleotide chemistries.

In addition, several approaches for designing and predicting efficiencyof specific oligonucleotides using an in vitro system were alsopublished (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].

Another agent capable of down-regulating RTVP-1 is a ribozyme moleculecapable of specifically cleaving an mRNA transcript encoding RTVP-1.Ribozymes are being increasingly used for the sequence-specificinhibition of gene expression by the cleavage of mRNAs encoding proteinsof interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. Thepossibility of designing ribozymes to cleave any specific target RNA hasrendered them valuable tools in both basic research and therapeuticapplications.

An additional method of regulating the expression of a RTVP-1 gene incells is via triplex forming oligonuclotides (TFOs). Recent studies haveshown that TFOs can be designed which can recognize and bind topolypurine/polypirimidine regions in double-stranded helical DNA in asequence-specific manner. These recognition rules are outlined by MaherIII, L. J., et al., Science, 1989; 245:725-730; Moser, H. E., et al.,Science, 1987; 238:645-630; Beal, P. A., et al, Science, 1992;251:1360-1363; Cooney, M., et al., Science, 1988; 241:456-459; andHogan, M. E., et al., EP Publication 375408.

Modification of the oligonuclotides, such as the introduction ofintercalators and backbone substitutions, and optimization of bindingconditions (pH and cation concentration) have aided in overcominginherent obstacles to TFO activity such as charge repulsion andinstability, and it was recently shown that synthetic oligonucleotidescan be targeted to specific sequences (for a recent review see Seidmanand Glazer, J Clin Invest 2003; 112:487-94).

In general, the triplex-forming oligonucleotide has the sequencecorrespondence:

oligo 3′--A G G T duplex 5′--A G C T duplex 3′--T C G A

However, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch, BMC Biochem,2002, Sep. 12, Epub). The same authors have demonstrated that TFOsdesigned according to the A-AT and G-GC rule do not form non-specifictriplexes, indicating that the triplex formation is indeed sequencespecific.

Triplex-forming oligonucleotides preferably are at least 15, morepreferably 25, still more preferably 30 or more nucleotides in length,up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs,and formation of the triple helical structure with the target DNAinduces steric and functional changes, blocking transcription initiationand elongation, allowing the introduction of desired sequence changes inthe endogenous DNA and resulting in the specific down-regulation of geneexpression. Examples of such suppression of gene expression in cellstreated with TFOs include knockout of episomal supFG 1 and endogenousHPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27:1176-81, and Puri, et al, J Biol Chem, 2001; 276:28991-98), and thesequence- and target specific down-regulation of expression of the Ets2transcription factor, important in prostate cancer etiology (Carbone, etal, Nucl Acid Res. 2003; 31:833-43) and the pro-inflammatory ICAM-1 gene(Besch et al, J Biol Chem, 2002; 277:32473-79). In addition, Vuyisichand Beal have recently shown that sequence specific TFOs can bind todsRNA, inhibiting activity of dsRNA-dependent enzymes such asRNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

Additionally, TFOs designed according to the abovementioned principlescan induce directed mutagenesis capable of effecting DNA repair, thusproviding both down-regulation and up-regulation of expression ofendogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94).Detailed description of the design, synthesis and administration ofeffective TFOs can be found in U.S. Patent Application Nos. 2003 017068and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 toEmanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.

The invention also contemplates silencing RTVP-1 by genetic modificationof the RTVP-1 locus. This modification can include complete deletion ofpart or all of the coding region such that no functional protein isproduced. Modification can also include mutation or deletion of the partor all of the promotor, such that the coding region is not transcribed.In some embodiments, silencing of RTVP-1 comprises introduction ofCRISPR/Cas9 reagents to genetically delete and/or modify the RTVP-1genomic locus.

The conditions used for contacting the mesenchymal stem cells areselected for a time period/concentration of cells/concentration ofRTVP-1 down-regulatory agent/ratio between cells and RTVP-1down-regulatory agent which enable the RTVP-1 down-regulatory agent toinduce differentiation thereof.

Isolated cell populations obtained according to the methods describeherein are typically non-homogeneous, although homogeneous cellpopulations are also contemplated.

According to a particular embodiment, the cell populations aregenetically modified to express an exogenous miRNA or a polynucleotideagent capable of down-regulating the miRNA.

The term “isolated” as used herein refers to a population of cells thathas been removed from its in-vivo location (e.g. bone marrow, neuraltissue). Preferably the isolated cell population is substantially freefrom other substances (e.g., other cells) that are present in itsin-vivo location.

Cell populations may be selected such that more than about 50%(alternatively more than about 60%, more than about 70%, more than about80%, more than about 90% or even more than about 95%) of the cellsexpress at least one, at least two, at least three, at least four, atleast five of the markers for motor neurons or at least one, at leasttwo, at least three, at least four, at least five of the markers forneural stem cells.

Isolation of particular subpopulations of cells may be affected usingtechniques known in the art including fluorescent activated cell sortingand/or magnetic separation of cells.

The cells of the populations of this aspect of the present invention maycomprise structural motor neuron or neural stem cell phenotypesincluding a cell size, a cell shape, an organelle size and an organellenumber. These structural phenotypes may be analyzed using microscopictechniques (e.g. scanning electro microscopy). Antibodies or dyes may beused to highlight distinguishing features in order to aid in theanalysis.

The cells and cell populations of the present invention may be usefulfor a variety of therapeutic purposes. Representative examples of CNSdiseases or disorders that can be beneficially treated with the cellsdescribed herein include, but are not limited to, a pain disorder, amotion disorder, a dissociative disorder, a mood disorder, an affectivedisorder, a neurodegenerative disease or disorder, psychiatric disordersand a convulsive disorder.

More specific examples of such conditions include, but are not limitedto, Parkinson's, ALS, Multiple Sclerosis, Huntingdon's disease,autoimmune encephalomyelitis, spinal cord injury, cerebral palsy,diabetic neuropathy, glaucatomus neuropathy, macular degeneration,action tremors and tardive dyskinesia, panic, anxiety, depression,alcoholism, insomnia, manic behavior, schizophrenia, autism-spectrumdisorder, manic-depressive disorders, Alzheimer's and epilepsy.

The use of differentiated MSCs may be also indicated for treatment oftraumatic lesions of the nervous system including spinal cord injury andalso for treatment of stroke caused by bleeding or thrombosis orembolism because of the need to induce neurogenesis and provide survivalfactors to minimize insult to damaged neurons.

The motor neuron like cells of the present invention may be useful formotor neuron diseases including, but not limited to amyotrophic lateralsclerosis (ALS), primary lateral sclerosis (PLS), pseudobulbar palsy andprogressive bulbar palsy.

In any of the methods described herein the cells may be obtained from anautologous, semi-allogeneic or non-autologous (i.e., allogeneic orxenogeneic) human donor or embryo or cord/placenta. For example, cellsmay be isolated from a human cadaver or a donor subject.

The term semi-allogeneic refers to donor cells which arepartially-mismatched to recipient cells at a major histocompatibilitycomplex (MHC) class I or class II locus.

The cells of the present invention can be administered to the treatedindividual using a variety of transplantation approaches, the nature ofwhich depends on the site of implantation.

The term or phrase “transplantation”, “cell replacement” or “grafting”are used interchangeably herein and refer to the introduction of thecells of the present invention to target tissue. As mentioned, the cellscan be derived from the recipient or from an allogeneic, semi-allogeneicor xenogeneic donor.

The cells can be injected systemically into the circulation,administered intrathecally or grafted into the central nervous system,the spinal cord or into the ventricular cavities or subdurally onto thesurface of a host brain. Conditions for successful transplantationinclude: (i) viability of the implant; (ii) retention of the graft atthe site of transplantation; and (iii) minimum amount of pathologicalreaction at the site of transplantation. Methods for transplantingvarious nerve tissues, for example embryonic brain tissue, into hostbrains have been described in: “Neural grafting in the mammalian CNS”,Bjorklund and Stenevi, eds. (1985); Freed et al., 2001; Olanow et al.,2003). These procedures include intraparenchymal transplantation, i.e.within the host brain (as compared to outside the brain orextraparenchymal transplantation) achieved by injection or deposition oftissue within the brain parenchyma at the time of transplantation.

Intraparenchymal transplantation can be performed using two approaches:(i) injection of cells into the host brain parenchyma or (ii) preparinga cavity by surgical means to expose the host brain parenchyma and thendepositing the graft into the cavity.

Both methods provide parenchymal deposition between the graft and hostbrain tissue at the time of grafting, and both facilitate anatomicalintegration between the graft and host brain tissue. This is ofimportance if it is required that the graft becomes an integral part ofthe host brain and survives for the life of the host.

Alternatively, the graft may be placed in a ventricle, e.g. a cerebralventricle or subdurally, i.e. on the surface of the host brain where itis separated from the host brain parenchyma by the intervening pia materor arachnoid and pia mater. Grafting to the ventricle may beaccomplished by injection of the donor cells or by growing the cells ina substrate such as 3% collagen to form a plug of solid tissue which maythen be implanted into the ventricle to prevent dislocation of thegraft. For subdural grafting, the cells may be injected around thesurface of the brain after making a slit in the dura. Injections intoselected regions of the host brain may be made by drilling a hole andpiercing the dura to permit the needle of a microsyringe to be inserted.The microsyringe is preferably mounted in a stereotaxic frame and threedimensional stereotaxic coordinates are selected for placing the needleinto the desired location of the brain or spinal cord. The cells mayalso be introduced into the putamen, nucleus basalis, hippocampuscortex, striatum, substantia nigra or caudate regions of the brain, aswell as the spinal cord.

The cells may also be transplanted to a healthy region of the tissue. Insome cases the exact location of the damaged tissue area may be unknownand the cells may be inadvertently transplanted to a healthy region. Inother cases, it may be preferable to administer the cells to a healthyregion, thereby avoiding any further damage to that region. Whatever thecase, following transplantation, the cells preferably migrate to thedamaged area.

For transplanting, the cell suspension is drawn up into the syringe andadministered to anesthetized transplantation recipients. Multipleinjections may be made using this procedure.

The cellular suspension procedure thus permits grafting of the cells toany predetermined site in the brain or spinal cord, is relativelynon-traumatic, allows multiple grafting simultaneously in severaldifferent sites or the same site using the same cell suspension, andpermits mixtures of cells from different anatomical regions.

Multiple grafts may consist of a mixture of cell types, and/or a mixtureof transgenes inserted into the cells. Preferably from approximately 104to approximately 109 cells are introduced per graft. Cells can beadministered concomitantly to different locations such as combinedadministration intrathecally and intravenously to maximize the chance oftargeting into affected areas.

For transplantation into cavities, which may be preferred for spinalcord grafting, tissue is removed from regions close to the externalsurface of the central nerve system (CNS) to form a transplantationcavity, for example as described by Stenevi et al. (Brain Res. 114:1-20,1976), by removing bone overlying the brain and stopping bleeding with amaterial such a gelfoam. Suction may be used to create the cavity. Thegraft is then placed in the cavity. More than one transplant may beplaced in the same cavity using injection of cells or solid tissueimplants. Preferably, the site of implantation is dictated by the CNSdisorder being treated. Demyelinated MS lesions are distributed acrossmultiple locations throughout the CNS, such that effective treatment ofMS may rely more on the migratory ability of the cells to theappropriate target sites.

Intranasal administration of the cells is also contemplated.

MSCs typically down regulate MHC class 2 and are therefore lessimmunogenic. Embryonal or newborn cells obtained from the cord blood,cord's Warton's gelly or placenta are further less likely to be stronglyimmunogenic and therefore less likely to be rejected, especially sincesuch cells are immunosuppressive and immunoregulatory to start with.

Notwithstanding, since non-autologous cells may induce an immunereaction when administered to the body several approaches have beendeveloped to reduce the likelihood of rejection of non-autologous cells.Furthermore, since diseases such as multiple sclerosis are inflammatorybased diseases, the problem of immune reaction is exacerbated. Theseinclude either administration of cells to privileged sites, oralternatively, suppressing the recipient's immune system, providinganti-inflammatory treatment which may be indicated to control autoimmunedisorders to start with and/or encapsulating thenon-autologous/semi-autologous cells in immunoisolating, semipermeablemembranes before transplantation.

As mentioned herein above, the present inventors also propose use ofcord and placenta-derived MSCs that express very low levels of MHCIImolecules and therefore limit immune response.

The following experiments may be performed to confirm the potential useof newborn's MSCs isolated from the cord I placenta for treatment ofneurological disorders:

1) Differentiated MSCs (to various neural cells or neural progenitorcells) may serve as stimulators in one-way mixed lymphocyte culture withallogeneic T-cells and proliferative responses in comparison with Tcells responding against allogeneic lymphocytes isolated from the samedonor may be evaluated by 3H Thymidine uptake to documenthyporesponsiveness.

2) Differentiated MSCs may be added/co-cultured to one-way mixedlymphocyte cultures and to cell cultures with T cell mitogens(phytohemmaglutinin and concanavalin A) to confirm the immunosuppressiveeffects on proliferative responses mediated by T cells.

3) Cord and placenta cells cultured from Brown Norway rats (unmodifiedand differentiated), may be enriched for MSCs and these cells may beinfused into Lewis rats with induced experimental autoimmuneencephalomyelitis (EAE). Alternatively, cord and placenta cells culturedfrom BALB/c mice, (BALB/cxC57BL/6)F1 or xenogeneic cells from BrownNorway rats (unmodified and differentiated), may be enriched for MSCsand these cells may be infused into C57BL/6 or SJL/j recipients withinduced experimental autoimmune encephalomyelitis (EAE). The clinicaleffects against paralysis may be investigated to evaluate thetherapeutic effects of xenogeneic, fully MHC mismatched orhaploidentically mismatched MSCs. Such experiments may provide the basisfor treatment of patients with a genetic disorder or genetically proneddisorder with family member's haploidentical MSCs.

4) BALB/c MSCs cultured from cord and placenta may be transfused withpre-miR labeled with GFP or RFP, which will allow the inventors tofollow the migration and persistence of these cells in the brain ofC57BL/6 recipients with induced EAE. The clinical effects of labeled MHCmismatched differentiated MSCs may be evaluated by monitoring signs ofdisease, paralysis and histopathology. The migration and localization ofsuch cells may be also monitored by using fluorescent cells fromgenetically transduced GFP “green” or Red2 “red” donors.

As mentioned, the present invention also contemplates encapsulationtechniques to minimize an immune response.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu M Z, et al., Cell encapsulation withalginate and alpha phenoxycinnamylidene-acetylated poly(allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol. Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., Anovel cell encapsulation method using photosensitive poly(allylaminealpha-cyanocinnamylideneacetate). J. Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a per-polymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 um. Such microcapsules can be furtherencapsulated with additional 2-5 um per-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesTechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, the quality control, mechanical stability,diffusion properties, and in vitro activities of encapsulated cellsimproved when the capsule size was reduced from 1 mm to 400 um (CanapleL. et al., Improving cell encapsulation through size control. J BiomaterSci Polym Ed. 2002; 13:783-96). Moreover, nanoporous biocapsules withwell-controlled pore size as small as 7 nm, tailored surface chemistriesand precise microarchitectures were found to successfully immunoisolatemicroenvironments for cells (Williams D. Small is beautiful:microparticle and nanoparticle technology in medical devices. Med DeviceTechnol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology forpancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

Examples of immunosuppressive agents include, but are not limited to,methotrexate, cyclophosphamide, cyclosporine, cyclosporin A,chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine),gold salts, D-penicillamine, leflunomide, azathioprine, anakinra,infliximab (REMICADE™), etanercept, TNF alpha blockers, a biologicalagent that targets an inflammatory cytokine, and Non-SteroidalAnti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are notlimited to acetyl salicylic acid, choline magnesium salicylate,diflunisal, magnesium, salicylate, salsalate, sodium salicylate,diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin,ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone,phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen,Cox-2 inhibitors and tramadol.

In any of the methods described herein, the cells can be administeredeither per se or, preferably as a part of a pharmaceutical compositionthat further comprises a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the cell compositions described herein, with otherchemical components such as pharmaceutically suitable carders andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of the cells to a subject.

Hereinafter, the term “pharmaceutically acceptable earlier” refers to acarrier or a diluent that does not cause significant irritation to asubject and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare propylene glycol, saline, emulsions and mixtures of organic solventswith water.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration include direct administration into thecirculation (intravenously or intra-arterial), into the spinal fluid orinto the tissue or organ of interest. Thus, for example the cells may beadministered directly into the brain.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. Preferably, a dose is formulated in ananimal model to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. For example, animal models ofdemyelinating diseases include shiverer (shi/shi, MBP deleted) mouse, MDrats (PLP deficiency), Jimpy mouse (PLP mutation), dog shaking pup (PLPmutation), twitcher mouse (galactosylceramidase defect, as in humanKrabbe disease), trembler mouse (PMP-22 deficiency). Virus induceddemyelination model comprise use if Theiler's virus and mouse hepatitisvirus. Autoimmune EAE is a possible model for multiple sclerosis.

The data obtained from these in vitro and cell culture assays and animalstudies can be used in formulating a range of dosage for use in human.The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition, (see e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1). For example, amultiple sclerosis patient can be monitored symptomatically for improvedmotor functions indicating positive response to treatment.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer.

Dosage amount and interval may be adjusted individually to levels of theactive ingredient which are sufficient to effectively treat the braindisease/disorder. Dosages necessary to achieve the desired effect willdepend on individual characteristics and route of administration.Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks ordiminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the individual being treated, the severity of theaffliction, the manner of administration, the judgment of theprescribing physician, etc. The dosage and timing of administration willbe responsive to a careful and continuous monitoring of the individualchanging condition. For example, a treated multiple sclerosis patientwill be administered with an amount of cells which is sufficient toalleviate the symptoms of the disease, based on the monitoringindications.

The cells of the present invention may be co-administered withtherapeutic agents useful in treating neurodegenerative disorders, suchas gangliosides; antibiotics, neurotransmitters, neurohormones, toxins,neurite promoting molecules; and antimetabolites and precursors ofneurotransmitter molecules such as L-DOPA.

As used herein the term “about” refers to +/−10%.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals there between.

It is noted that for each miR described herein the correspondingsequence (mature and pre) is provided in the sequence listing whichshould be regarded as part of the specification.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in anon-limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cclls-e-A Manual of BasicTechnique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition;“Current Protocols in Immunology” Volumes I-III Coligan J. E., ed.(1994); Stites et al. (eds), “Basic and Clinical Immunology” (8thEdition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi(eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co.,New York (1980); available immunoassays are extensively described in thepatent and scientific literature, see, for example, U.S. Pat. Nos.3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517;3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074;4,098,876; 4,879,219; 5,011,771 and 5,281,521; “OligonucleotideSynthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames,B. D., and Higgins S. J., eds. (1985); “Transcription and Translation”Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture”Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press,(1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and“Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: AGuide To Methods And Applications”, Academic Press, San Diego, Calif.(1990); Marshak et al., “Strategies for Protein Purification andCharacterization=—A Laboratory Course Manual” CSHL Press (1996); all ofwhich are incorporated by reference as if fully set forth herein. Othergeneral references are provided throughout this document. The procedurestherein are believed to be well known in the art and are provided forthe convenience of the reader. All the information contained therein isincorporated herein by reference.

Example 1 Differentiation of Mesenchymal Stem Cells (MSCs) to NeuralStem Cells (NSCs)

Methods

Mesenchymal stem cells (MSCs) from either bone marrow, adipose, placentaor umbilical cord were plated in high density in bacterial dishes inserum free medium supplemented with 10 mg/ml EGF and bFGF for 10 days.The cells started to aggregate and after 4-5 days were disaggregatedmechanically to promote their detachment from the plates. The cells werethen maintained for two weeks after which they were analyzed for theexpression of NSC markers and for their ability to generate neurons,astrocytes and oligodendrocytes when plated on laminin in low-serum (5%)medium.

The cells were then subjected to miRNA microarray as described.

Results

As illustrated in FIGS. 1A-B, the mesenchymal stem cells expressedneuronal markers following neural stem cell differentiation.

Example 2 Changes in miRNA Expression During NSC Differentiation

Materials and Methods

miRNAs have been shown to play a role in the differentiation of variousneural cells and neural stem cells. To analyze the expression andfunction of specific miRNAs in MSC-derived NSCs, the MSCs weredifferentiated towards NSCs as described in Example 1 and miRNA arrayanalysis was performed to the control and differentiated cells. AqRT-PCR microarray was run that contained 96 miRNAs, all of which wererelated to stem cells and that were divided into subgroups based ontheir known association with stem cells, neural-related, hematopoieticand organ-related miRNAs.

For analyzing the differential expression of specific miRNA in controland differentiated MSCs, the Stem cell microRNA qPCR array was employedwith quantiMiR from SBI company (catalog # RA620A-1), according to theuser protocol, the contents of which are incorporated herein byreference. For the qPCR, the Applied Biosystems Power SYBR master mix(cat #4367659) was used.

The system allows for the ability to quantitate fold differences of 95separate microRNAs between 2 separate experimental RNA samples. Thearray plate also includes the U6 transcript as a normalization signal.All 95 microRNAs chosen for the array have published implications withregard to potential roles in stem cell self-renewal, hematopoiesis,neuronal development and differentiated tissue identification.

The array plate also includes the U6 RNA as a normalization signal.

Total RNA was isolated from 105-106 cells of control and differentiatedMSCs using miRneasy total RNA isolation kit from Qiagen (catalog#217004) that isolate RNA fraction with sizes <200 bp. 500 ng of totalRNA was processed according to “SBI Stem Cell MicroRNA qPCR Array withQuantiMir™” (Cat. # RA620A-1) user protocol. For the qPCR, the AppliedBiosystems Power SYBR master mix (cat #4367659) was used.

For validation, sybr-green qPCR of the specific miRNA of interest wasperformed on the same RNA samples processed according to QIAGEN miScriptSystem handbook (cat #218061 & 218073) Hu hsa-miR MicroRNA Profiling Kit(System Biosciences) “SBI Stem Cell MicroRNA qPCR Array with QuantiMir™”(Cat. # RA620A-1) which detects the expression of 96 miRNAs, was used toprofile the miRNAs in unmodified BM-MSC compared with MSCsdifferentiated to astrocytes. 500 ng of total RNA was tagged withpoly(A) to its 3′ end by poly A polymerase, and reverse-transcribed witholigo-dT adaptors by QuantiMir RT technology. Expression levels of themiRNAs were measured by quantitative PCR using SYBR green reagent andVIIA7, Real-Time PCR System (Applied Biosystems). All miRNAs could bemeasured with miRNA specific forward primers and a universal reverseprimer (SBI). Expression level of the miRNAs was normalized to U6 snRNA,using the comparative CT method for relative quantification ascalculated with the following equation: 2-[(CT astrocyte diff miRNA-CTastrocyte endogenous control)-(CTDMEM miRNA-CT DMEM endogenous control).

In addition, an Affymetrix miRNA 3.0 array was used to compare BM-MSCsand human NSCs and identify differentially expressed miRNAs.

Results

As presented in FIGS. 2, 3 and 4A, there were significant changes in theexpression of specific miRNA of each group between the control MSCs andthe differentiated ones.

The results of the Affymetrix miRNA 3.0 array analysis are detailed inTable 1 herein below.

Using a nestin promoter based reporter assay, the present inventorsconfirmed that overexpression of miR-20b, miR-935, miR-891 and miR-378also induced differentiation of the MSCs into NSCs (FIG. 4B).

Similarly, silencing of miR-138, miR-214, miR-199a and miR-199bdecreased the mesenchymal phenotypes of all the MSCs and induced theirNSC differentiation (FIG. 4C).

Co-transfection of the MSCs with combination of miR-20b or miR-378 withantagomiR-138 further increased the differentiation of the MSCs tonestin positive cells (FIG. 4D).

As presented in FIGS. 4E-F, overexpression of antagomiR-138 and miR-891mimic induced a significant increase in the generation of nestinpositive cells in the transfected MSCs as demonstrated by the increasedfluorescence intensity of cells transduced with the nestin-GFP reporter.

TABLE 1 Up Down MSCs/NSCs regulated MSCs/NSCs regulated miRNA Foldchange miRNA Fold change miRNA Fold change hsa-miR- 1379.78 hsa-let-−1.53698 hsa-miR- −7.34456 145_st 7c_st 324-3p_st hsa-miR- 752.7381hsa-miR- −1.58884 hsa-miR- −7.83858 143_st 665_st 20a_st hsa-miR-552.6854 hsa-miR- −1.61841 hsa-miR- −8.36351 214_st 4258_st 501-5p_sthsa-miR- 511.1263 hsa-miR- −1.63684 hsa-miR- −8.71869 199a-3p_st361-3p_st 330-3p_st hsa-miR- 362.5667 hsa-miR- −1.76218 hsa-miR-−9.13392 199a-5p_st 374a-star_st 874_st hsa-miR- 347.4311 hsa-miR-−1.85672 hsa-miR- −9.68441 199b-3p_st 892b_st 500_st hsa-miR- 229.2463hsa-miR- −1.90874 hsa-miR- −9.86881 138_st 361-5p_st 25_st hsa-miR-190.5331 hsa-miR- −1.93941 hsa-miR- −10.1382 31_st 181a_st 769-5p_sthsa-miR- 59.83459 hsa-miR- −2.19583 hsa-miR- −10.3325 21_st 16_st125b-2-star_st hsa-miR- 23.8986 hsa-miR- −2.27398 hsa-miR- −16.7436193a-5p_st 636_st 130b_st hsa-miR- 21.60842 hsa-miR- −2.79417 hsa-miR-−16.9435 224-star_st 4284_st 504_st hsa-miR- 21.38142 hsa-miR- −3.00768hsa-miR- −17.7877 196a_st 1208_st 181a-2-star_st hsa-miR- 19.18475hsa-miR- −3.01855 hsa-miR- −20.1501 487b_st 1274b_st 885-3p_st hsa-miR-17.45522 hsa-miR- −3.46182 hsa-miR- −21.0971 409-5p_st 30c-2-star_st1246_st hsa-miR- 10.34438 hsa-miR- −3.49025 hsa-miR- −22.8735193b-star_st 501-3p_st 92b_st hsa-miR- 9.571106 hsa-miR- −3.7152hsa-miR- −23.3686 379_st 92a_st 362-5p_st hsa-miR- 8.401508 hsa-miR-−3.72739 hsa-miR- −23.3743 21-star_st 378b_st 572_st hsa-miR- 7.080883hsa-miR- −3.87466 hsa-miR- −24.4173 27a-star_st 1287_st 4270_st hsa-miR-6.122331 hsa-miR- −4.0524 hsa-miR- −26.6758 27a_st 425-star_st 378c_sthsa-miR- 5.715753 hsa-miR- −4.37339 hsa-miR- −28.4948 4317_st 324-5p_st93-star_st hsa-miR- 4.920511 hsa-miR- −4.40631 hsa-miR- −28.7369 193b_st3178_st 149_st hsa-miR- 4.889609 hsa-miR- −4.52146 hsa-miR- −28.996827b_st 219-1-3p_st 363_st hsa-miR- 4.798265 hsa-miR- −4.609 hsa-miR-−31.2283 22_st 197_st 9_st hsa-miR- 3.402782 hsa-miR- −4.61406 hsa-miR-−32.3908 574-3p_st 181b_st 18a_st hsa-miR- 3.375774 hsa-miR- −4.72807hsa-miR- −33.1912 4288_st 500-star_st 891a_st hsa-miR- 3.34163 hsa-miR-−4.96582 hsa-miR- −38.7283 23a_st 106b_st 346_st hsa-miR- 3.09015hsa-miR- −4.97984 hsa-miR- −50.7583 221-star_st 502-3p_st 124_sthsa-miR- 3.030064 hsa-miR- −5.17107 hsa-miR- −72.2314 2113_st 30c_st497_st hsa-let- 2.551577 hsa-miR- −5.29365 hsa-miR- −73.6306 7i_st1275_st 378_st hsa-miR- 2.300083 hsa-miR- −5.54416 hsa-miR- −82.706624_st 422a_st 1231_st hsa-miR- 2.217338 hsa-miR- −5.6233 hsa-miR-−92.6078 23b_st 93_st 139-5p_st hsa-miR- 2.201907 hsa-miR- −5.74741hsa-miR- −94.3695 299-3p_st 181d_st 3180-3p_st hsa-miR- 2.197822hsa-miR- −5.82664 hsa-miR- −114.107 518c-star_st 1307_st 9-star_sthsa-miR- 2.186328 hsa-miR- −5.84397 hsa-miR- −140.688 221_st 1301_st935_st hsa-miR- 2.177192 hsa-miR- −5.88481 hsa-miR- −156.762 431-star_st99a_st 20b_st hsa-miR- 2.116276 hsa-miR- −5.9383 523_st 505-star_sthsa-miR- 1.937531 hsa-miR- −5.94177 4313_st 1202_st hsa-miR- 1.916531hsa-miR- −6.05212 559_st 128_st hsa-miR- 1.894046 hsa-miR- −6.11976614_st 532-5p_st hsa-miR- 1.803374 hsa-miR- −6.5161 653_st 195_sthsa-miR- 1.675887 hsa-miR- −6.66014 2278_st 532-3p_st v11_hsa-miR-1.647103 hsa-miR- −6.91155 768-5p_st 106a_st hsa-miR- 1.608659 hsa-miR-−6.91565 154-star_st 17_st hsa-miR- 1.598961 hsa-miR- −7.05548302a-star_st 1271_st hsa-miR- 1.580479 hsa-miR- −7.1367 3199_st769-3p_st hsa-miR- 1.476948 hsa-miR- −7.31636 3137_st 15b_st

Example 3 miRNAs that Play a Role in the Differentiation of MSCs to NSCs

The present inventors further examined the role of the specific miRNAsthat were found to be altered in the miR microarray on thedifferentiation of the MSCs to NSCs. These experiments were performed bytransfecting MSCs with either specific or combination of mature miRNAmimics or miRNA inhibitors and then their ability to generateneurospheres and express the markers nestin and Sox2 was examined.

Results

It was found that the inhibition of let-7 together with expression ofmiR-124 increased NSC differentiation.

In addition, it was found that up-regulation of the following miRNAs:miR302b, miR-371, miR-134, miR-219, miR-154, miR-155, miR-32, miR-33,miR-126 and miR-127 and down-regulation of the following miRs-miR-10b,miR-142-3p, miR-131a, miR-125b, miR-153 and miR-181a either alone or invarious combinations induced differentiation of the MSCs to NSCs albeitto different degrees.

In addition to the miRNAs that were described in the miRNA array, it wasalso found that transfection of the MSCs with miR-132 and miR-137 alsoincreased the NSC differentiation.

Example 4 Additional Factors that Promote the Differentiation of MSCs toNSCs

Related to testis-specific, vespid and pathogenesis protein 1 (RTVP-1)was cloned from human GBM cell lines by two groups and was termed gliomapathogenesis-related protein-GLIPR1 or RTVP-1 [3]. RTVP-1 contains aputative signal peptide, a transmembrane domain and a SCP domain, with ayet unknown function which is also found in other RTVP-1 homologsincluding TPX-1 [4], the venom allergen antigen 5 [5] and group 1 of theplant pathogenesis-related proteins (PR-1). It has recently beenreported that RTVP-1 acts as a tumor promoter in gliomas. Thus, theexpression of RTVP-1 correlates with the degree of malignancy ofastrocytic tumors and over-expression of RTVP-1 increases cellproliferation, invasion, migration and anchorage independent growth.Moreover, silencing of RTVP-1 induces apoptosis in glioma cell lines andprimary glioma cultures [6]. Interestingly, RTVP-1 acts as a tumorsuppressor in prostate cancer cells and adenovirus mediated delivery ofRTVP-1 has therapeutic effects in a mouse prostate cancer model [7-9].

Results

Expression of RTVP-1 in MSCs is very high, as determined by Western blot(FIG. 5A). Moreover, silencing of RTVP-1 in MSCs abrogated their abilityto differentiate to mesenchymal lineage cells (FIGS. 5C-D).

Further, silencing of RTVP-1 in MSCs increased the expression of bothnestin and Sox 2 and some levels of beta 3 tubulin (data not shown).Interestingly, it was found that RTVP-1 is a novel target of miR-137,suggesting that the positive effect of miR-137 on the NSCdifferentiation of MSCs may be mediated by RTVP-1.

To further examine the role of RTVP-1, its expression was examined inMSCs, NSCs and in MSCs that were differentiated into NSCs. Human NSCsdid not express RTVP-1 at all (data not shown) and the expression ofRTVP-1 in MSCs was significantly higher than that of MSCs differentiatedto NSCs irrespective of the source of MSCs that were examined (FIG. 5E).

The effect of RTVP-1 overexpression in human NSCs was examined. It wasfound that these cells acquired mesenchymal phenotypes and especiallywere predisposed to differentiate into adipocytes (data not shown).

Silencing of RTVP-1 in the different MSCs examined increased theexpression of nestin in these cells (FIG. 5F).

To further analyze the effect of RTVP-1 on mesenchymal transformation,gene array analysis was performed on BM-MSCs in which the expression ofRTVP-1 was silenced. Silencing of RTVP-1 decreased the expression ofALDH1A3 by 3.2-fold, VAV3 by 15 fold, CD200 by 5 fold and the sternnessmarkers Oct4, Nanog and Sox2 by 2.3, 3.4 and 4.2, respectively.Collectively these results indicate that RTVP-1 decreases theproliferation and sternness signature of these cells.

In contrast, RTVP-1 silencing increased the expression of certain genessuch as nestin (3.4 fold), NKX2.2 (4.7 fold) and calcium channel,voltage dependent (3 fold).

Together, these results implicate RTVP-1 as a major mesenchymalregulator and demonstrate that silencing of RTVP-1 inducesdifferentiation of MSCs to cells with neural phenotypes.

Example 5 Differentiation of Neural Progenitor Cells to Motor Neurons

Materials and Methods

Plates were coated with 20 μg/ml laminin overnight and were then washedtwice with PBS. The NPC were plated in the confluency of 50% and after24 hr were incubated with priming medium: NM medium with heparin (use 10μg/mL) and bFGF (100 μg/mL) for 5 days. After day 5 the medium waschanged to the differentiation medium: F12 with 1 mL of B27 in 50 mL F12(or 2%), retinoic acid (RA, 1 μM), and SHH (200 ng/mL). The RA was addedevery other day. After 5 days GDNF and BDNF were added to the medium (10ng/mL).

Results

In the developing spinal cord, there is sequential generation of motorneurons (MNs) and oligodendrocytes (OLPs). There are common progenitorscalled pMN that first generate MN and then oligodendrocytes. The basichelix-loop-helix (bHLH) transcription factor Olig2, is expressed in thepMN domain and it's one of the important transcription factors that playa role in the development of both cell types. Over-expression of Olig2in MSCs that were grow in NM medium supplemented with 200 ng/mlrecombinant SHH, 20 ng/ml of each, GDNF, BDNF, CNTF and NT-3 and 1 mMretinoic acid induced the expression of two specific markers of motorneurons Hb9 and Islet1 (FIGS. 6A-D).

Example 6 Involvement of miRNAs in the Differentiation of NPCs to MotorNeurons

Materials and Methods

To identify specific miRNAs involved with motor neuron differentiation,the present inventors differentiated two types of neural stem/progenitorcells into motor neurons at different stages of development using theprotocol described in Example 5. The characterization of the cells asmotor neurons was characterized by the expression of the specificmarkers, islet1, HB9 and the neuronal markers neurofilament and tubulin.

To analyze the expression and function of specific miRNAs in motorneurons the neural progenitor cell system described herein above wasused. miRNA array analysis was performed on the control anddifferentiated cells. A qRT-PCR microarray that contained 96 miRNAs, allof which were related to stem cells and that were divided into subgroupsbased on their known association with stem cells, neural-related,hematopoietic and organ-related miRNAs, as described in Example 2.

Results

As illustrated in FIGS. 7A-B, neural stem cells may be induced todifferentiate into motor neurons.

As presented in FIGS. 8-10, there were significant changes in theexpression of specific miRNA of each group between the control MSCs andthe differentiated MSCs.

qRT-PCR studies were performed to validate the differences in the miRNAexpression that were observed between the control and differentiatedcells.

Similar to the results that were obtained with the microarray data, theqRT-PCR results demonstrated a decrease in miRs, 372, 373, 141, 199a,32, 33, 221 and 223.

In contrast a significant increase was observed in all the miRNAs thatincreased in the array and specifically the following miRNAs: miR-368,302b, 365-3p, 365-5p, Let-7a, Let-7b, 218, 134, 124, 125a, 9, 154, 20a,130a.

The present inventors further examined the role of the specific miRNAsin the differentiation of MSCs to motor neurons. It was found that thecombination of Let-7a and miR-124, 368 and miR-154 increased theexpression of Hb9 and Islet-1. Similarly, transfection with combinationsof miR-125a, 9, 130a and 218, 134 and 20a together and in combinationwith miRNA inhibitors of miR-141, 32, 33, 221, 223 and miR373 alsoinduced differentiation of MSCs to either motor neuron progenitors or toimmature motor neurons.

Example 7 Sequences

TABLE 2 Sequence of Sequence of Name mature miRNA premiRNA hsa-let-7aseq id no: 1 seq id no: 73 seq id no: 74 seq id no: 75 hsa-let-7b seq idno: 2 seq id no: 76 hsa-let-7c seq id no: 3 seq id no: 77 hsa-let-7d seqid no: 4 seq id no: 78 hsa-let-7e seq id no: 5 seq id no: 79 hsa-let-7fseq id no: 6 seq id no: 80 hsa-let-7g seq id no: 7 seq id no: 81hsa-let-7i seq id no: 8 seq id no: 82 hsa-mir-106a seq id no: 9 seq idno: 83 hsa-mir-106b seq id no: 10 seq id no: 84 hsa-mir-1294 seq id no:11 seq id no: 85 hsa-mir-1297 seq id no: 12 seq id no: 86 hsa-mir-143seq id no: 13 seq id no: 87 hsa-mir-144 seq id no: 14 seq id no: 88hsa-mir-145 seq id no: 15 seq id no: 89 hsa-mir-17 seq id no: 16 seq idno: 90 miR-181a seq id no: 17 seq id no: 91 miR-181a seq id no: 18 seqid no: 92 miR-181b seq id no: 19 seq id no: 93 miR-181b seq id no: 20seq id no: 94 miR-181c seq id no: 21 seq id no: 95 hsa-mir-181d seq idno: 22 seq id no: 96 hsa-mir-199a-3p seq id no: 23 seq id no: 97hsa-mir-199b-3p seq id no: 24 seq id no: 98 hsa-mir-202 seq id no: 25seq id no: 99 hsa-mir-20a seq id no: 26 seq id no: 100 hsa-mir-20b seqid no: 27 seq id no: 101 hsa-mir-2113 seq id no: 28 seq id no: 102hsa-mir-25 seq id no: 29 seq id no: 103 hsa-mir-26a seq id no: 30 seq idno: 104 seq id no: 31 seq id no: 105 hsa-mir-26b seq id no: 32 seq idno: 106 hsa-mir-29a seq id no: 33 seq id no: 107 hsa-mir-29b seq id no:34 seq id no: 108 seq id no: 109 hsa-mir-29c seq id no: 35 seq id no:110 hsa-mir-3129-5p seq id no: 36 seq id no: 111 hsa-mir-3177-5p seq idno: 37 seq id no: 112 hsa-mir-32 seq id no: 38 seq id no: 113hsa-mir-326 seq id no: 39 seq id no: 114 hsa-mir-330-5p seq id no: 40seq id no: 115 hsa-mir-363 seq id no: 41 seq id no: 116 hsa-mir-3659 seqid no: 42 seq id no: 117 hsa-mir-3662 seq id no: 43 seq id no: 118hsa-mir-367 seq id no: 44 seq id no: 119 hsa-mir-372 seq id no: 45 seqid no: 120 hsa-mir-373 seq id no: 46 seq id no: 121 hsa-mir-3927 seq idno: 47 seq id no: 122 hsa-mir-4262 seq id no: 48 seq id no: 123hsa-mir-4279 seq id no: 49 seq id no: 124 hsa-mir-4458 seq id no: 50 seqid no: 125 hsa-mir-4465 seq id no: 51 seq id no: 126 hsa-mir-4500 seq idno: 52 seq id no: 127 hsa-mir-4658 seq id no: 53 seq id no: 128hsa-mir-4724-3p seq id no: 54 seq id no: 129 hsa-mir-4742-3p seq id no:55 seq id no: 130 hsa-mir-4770 seq id no: 56 seq id no: 131 hsa-mir-519dseq id no: 57 seq id no: 132 hsa-mir-520a-3p seq id no: 58 seq id no:133 hsa-mir-520b seq id no: 59 seq id no: 134 hsa-mir-520c-3p seq id no:60 seq id no: 135 hsa-mir-520d-3p seq id no: 61 seq id no: 136hsa-mir-520d-5p seq id no: 62 seq id no: 137 hsa-mir-520e seq id no: 63seq id no: 138 hsa-mir-524-5p seq id no: 64 seq id no: 139 hsa-mir-642bseq id no: 65 seq id no: 140 hsa-mir-656 seq id no: 66 seq id no: 141hsa-mir-767-5p seq id no: 67 seq id no: 142 hsa-mir-92a seq id no: 68seq id no: 143 seq id no: 69 seq id no: 144 hsa-mir-92b seq id no: 70seq id no: 145 hsa-mir-93 seq id no: 71 seq id no: 146 hsa-mir-98 seq idno: 72 seq id no: 147

TABLE 3 Sequence of Sequence of Name mature premiRNA hsa-mir-410 seq idno: 148 seq id no: 156 hsa-mir-3163 seq id no: 149 seq id no: 157hsa-mir-148a seq id no: 150 seq id no: 158 hsa-mir-148b seq id no: 151seq id no: 159 hsa-mir-152 seq id no: 152 seq id no: 160 hsa-mir-3121-3pseq id no: 153 seq id no: 161 hsa-mir-495 seq id no: 154 seq id no: 162hsa-mir-4680-3p seq id no: 155 seq id no: 163

TABLE 4 Sequence of Sequence of Name mature PMIR id premiRNA miR-92apseq id no: 164 MI0000093 seq id no: 269 seq id no: 165 MI0000094 seq idno: 270 miR-21 seq id no: 166 MI0000077 seq id no: 271 miR-26a 5P seq idno: 167 MI0000083 seq id no: 272 seq id no: 168 MI0000750 seq id no: 273miR-18a seq id no: 169 MI0000072 seq id no: 274 miR-124 seq id no: 170MI0000445 seq id no: 275 seq id no: 171 MI0000443 seq id no: 276 seq idno: 172 MI0000444 seq id no: 277 miR-99a seq id no: 173 MI0000101 seq idno: 278 miR-30c seq id no: 174 MI0000736 seq id no: 279 MI0000254 seq idno: 280 miR-301a 3P seq id no: 175 MI0000745 seq id no: 281 miR-145-50seq id no: 176 MI0000461 seq id no: 282 miR-143-3p seq id no: 177MI0000459 seq id no: 283 miR-373 3P seq id no: 178 MI0000781 seq id no:284 miR-20b seq id no: 179 MI0001519 seq id no: 285 miR-29c 3P seq idno: 180 MI0000735 seq id no: 286 miR-29b 3P seq id no: 181 MI0000105 seqid no: 287 MI0000107 seq id no: 288 miR-143 let-7g seq id no: 182MI0000433 seq id no: 289 let-7a seq id no: 183 MI0000060 seq id no: 290MI0000061 seq id no: 291 MI0000062 seq id no: 292 let-7b seq id no: 184MI0000063 seq id no: 293 miR-98 seq id no: 185 MI0000100 seq id no: 294miR-30a* seq id no: 186 MI0000088 seq id no: 295 miR-17 seq id no: 187MI0000071 seq id no: 296 miR-1-1 seq id no: 188 MI0000651 seq id no: 297miR-1-2 seq id no: 189 MI0000437 seq id no: 298 miR-192 seq id no: 190MI0000234 seq id no: 299 miR-155 seq id no: 191 MI0000681 seq id no: 300miR-516-ap a1- seq id no: 192 MI0003180 seq id no: 301 5p-- a2-3p-- seqid no: 193 MI0003181 seq id no: 302 miR-31 seq id no: 194 MI0000089 seqid no: 303 miR-181a seq id no: 195 MI0000289 seq id no: 304 seq id no:196 MI0000269 seq id no: 305 miR-181b seq id no: 197 MI0000270 seq idno: 306 seq id no: 198 MI0000683 seq id no: 307 miR-181c seq id no: 199MI0000271 seq id no: 308 miR-34-c seq id no: 200 MI0000743 seq id no:309 miR-34b* seq id no: 201 MI0000742 seq id no: 310 miR-103a seq id no:202 MI0000109 seq id no: 311 seq id no: 203 MI0000108 seq id no: 312miR-210 seq id no: 204 MI0000286 seq id no: 313 miR-16 seq id no: 205MI0000070 seq id no: 314 seq id no: 206 MI0000115 seq id no: 315 miR-30aseq id no: 207 MI0000088 seq id no: 316 miR-31 seq id no: 208 MI0000089seq id no: 317 miR-222 seq id no: 209 MI0000299 seq id no: 318 miR-17seq id no: 210 MI0000071 seq id no: 319 miR-17* seq id no: 211 MI0000071seq id no: 320 miR-200b seq id no: 212 MI0000342 seq id no: 321 miR-200cseq id no: 213 MI0000650 seq id no: 322 miR-128 seq id no: 214 MI0000447seq id no: 323 MI0000727 seq id no: 324 miR-503 seq id no: 215 MI0003188seq id no: 325 miR-424 seq id no: 216 MI0001446 seq id no: 326 miR-195seq id no: 217 MI0000489 seq id no: 327 miR-1256 seq id no: 218MI0006390 seq id no: 328 miR-203a seq id no: 219 MI0000283 seq id no:329 miR-199 ?? hsa-miR-199a- seq id no: 220 MI0000242 seq id no: 3303p_st hsa-miR-199a- seq id no: 221 MI0000242 seq id no: 331 5p_sthsa-miR-199b- seq id no: 222 MI0000282 seq id no: 332 3p_st miR-93 seqid no: 223 MI0000095 seq id no: 333 miR-98 seq id no: 224 MI0000100 seqid no: 334 miR-125-a seq id no: 225 MI0000469 seq id no: 335 miR-133aseq id no: 226 MI0000450 seq id no: 336 MI0000451 seq id no: 337miR-133b seq id no: 227 MI0000822 seq id no: 338 miR-126 seq id no: 228MI0000471 seq id no: 339 miR-194 seq id no: 229 MI0000488 seq id no: 340MI0000732 seq id no: 341 miR-346 seq id no: 230 MI0000826 seq id no: 342miR-15b seq id no: 231 MI0000438 seq id no: 343 miR-338-3p seq id no:232 MI0000814 seq id no: 344 miR-373 miR-205 seq id no: 233 MI0000285seq id no: 345 miR-210 miR-125 miR-1226 seq id no: 234 MI0006313 seq idno: 346 miR-708 seq id no: 235 MI0005543 seq id no: 347 miR-449 seq idno: 236 MI0001648 seq id no: 348 miR-422 seq id no: 237 MI0001444 seq idno: 349 miR-340 seq id no: 238 MI0000802 seq id no: 350 miR-605 seq idno: 239 MI0003618 seq id no: 351 miR-522 seq id no: 240 MI0003177 seq idno: 352 miR-663 seq id no: 241 MI0003672 seq id no: 353 miR-130a seq idno: 242 MI0000448 seq id no: 354 miR-130b seq id no: 243 MI0000748 seqid no: 355 miR-942 seq id no: 244 MI0005767 seq id no: 356 miR-572 seqid no: 245 MI0003579 seq id no: 357 miR-520 miR-639 seq id no: 246MI0003654 seq id no: 358 miR-654 seq id no: 247 MI0003676 seq id no: 359miR-519 miR-204 seq id no: 248 MI0000284 miR-224 seq id no: 249MI0000301 seq id no: 360 miR-616 seq id no: 250 MI0003629 seq id no: 361miR-122 seq id no: 251 MI0000442 seq id no: 362 miR-299 3p- seq id no:252 MI0000744 seq id no: 363 5p- seq id no: 253 seq id no: 364 miR-100seq id no: 254 MI0000102 miR-138 seq id no: 255 MI0000476 seq id no: 365miR-140 seq id no: 256 MI0000456 seq id no: 366 miR-375 seq id no: 257MI0000783 seq id no: 367 miR-217 seq id no: 258 MI0000293 seq id no: 368miR-302 seq id no: 369 miR-372 seq id no: 259 MI0000780 miR-96 seq idno: 260 MI0000098 seq id no: 370 miR-127-3p seq id no: 261 MI0000472 seqid no: 371 miR-449 seq id no: 372 miR-135b seq id no: 262 MI0000810miR-101 seq id no: 263 MI0000103 seq id no: 373 MI0000739 seq id no: 374miR-326 seq id no: 264 MI0000808 seq id no: 375 miR-3245p- seq id no:265 MI0000813 seq id no: 376 3p- seq id no: 266 MI0000813 seq id no: 377miR-335 seq id no: 267 MI0000816 seq id no: 378 miR-141 seq id no: 268MI0000457 seq id no: 379

TABLE 5 Sequence of Sequence of Name mature miRNA premiRNA miR-1275 seqid no: 381 seq id no: 414 miR-891a seq id no: 382 seq id no: 415 miR-154seq id no: 383 seq id no: 416 miR-1202 seq id no: 384 seq id no: 417miR-572 seq id no: 385 seq id no: 418 miR-935a seq id no: 386 seq id no:419 miR-4317 seq id no: 387 seq id no: 420 miR-153 seq id no: 388 seq idno: 421 seq id no: 422 miR-4288 seq id no: 389 seq id no: 423 miR-409-5pseq id no: 390 seq id no: 424 miR-193a-5p seq id no: 391 seq id no: 425miR-648 seq id no: 392 seq id no: 426 miR-368 miR-365 seq id no: 393 seqid no: 427 miR-500 seq id no: 394 seq id no: 428 miR-491 seq id no: 395seq id no: 429 hsa-miR-199a- seq id no: 396 seq id no: 430 3p_st seq idno: 397 seq id no: 431 hsa-miR-199a- seq id no: 398 seq id no: 432 5p_stseq id no: 399 seq id no: 433 miR-2113 seq id no: 400 seq id no: 434miR-372 seq id no: 401 seq id no: 435 miR-373 seq id no: 402 seq id no:436 miR-942 seq id no: 403 seq id no: 437 miR-1293 seq id no: 404 seq idno: 438 miR-18 seq id no: 405 seq id no: 439 miR-1182 seq id no: 406 seqid no: 440 miR-1185 seq id no: 407 seq id no: 441 seq id no: 442miR-1276 seq id no: 408 seq id no: 443 miR-193b seq id no: 409 seq idno: 444 miR-1238 seq id no: 410 seq id no: 445 miR-889 seq id no: 411seq id no: 446 miR-370 seq id no: 412 seq id no: 447 miR-548-d1 seq idno: 413 seq id no: 448

TABLE 6 mir designation seq id no: hsa-miR-302b seq id no: 449hsa-miR-371 seq id no: 450 hsa-miR-134 seq id no: 451 hsa-miR-219 seq idno: 452 hsa-miR-154 seq id no: 453 hsa-miR-155 seq id no: 454 hsa-miR-32seq id no: 455 hsa-miR-33 seq id no: 456 hsa-miR-126 seq id no: 457hsa-miR-127 seq id no: 458 hsa-miR-132 seq id no: 459 hsa-miR-137 seq idno: 460 hsa-miR-10b seq id no: 461 hsa-miR-142-3p seq id no: 462hsa-miR-131a hsa-miR-125b seq id no: 463 hsa-miR-153 seq id no: 464hsa-miR-181a seq id no: 465 hsa-miR-123 hsa-miR-let-7a seq id no: 466hsa-miR-let-7b seq id no: 467 hsa-miR-368 seq id no: 468 hsa-miR-365-3phsa-miR-365-5p hsa-miR-218 seq id no: 469 hsa-miR-124 seq id no: 470hsa-miR-125a seq id no: 471 hsa-miR-9 seq id no: 472 hsa-miR-20a seq idno: 473 hsa-miR-130a seq id no: 474 hsa-miR-372 seq id no: 475hsa-miR-373 seq id no: 476 hsa-miR-141 seq id no: 477 hsa-miR-199a seqid no: 478 hsa-miR-221 seq id no: 479 hsa-miR-223 seq id no: 480

Example 8 miRNAs that Play a Role in the Differentiation of MSCs toMotor Neurons

Materials and Methods

Plates were coated with 20 μg/ml laminin overnight and were then washedtwice with PBS. The MSCs were plated in the confluency of 50% and after24 hr were incubated with priming medium: NM medium with heparin (use 10μg/mL) and bFGF (10 μg/mL) for 5 days. After day 5 the medium waschanged to the differentiation medium: F12 with 1 mL of B27 in 50 μL F12(or 2%), retinoic acid (RA, 0.1-1 μM), and SHH (200 ng/mL). The RA wasadded every other day. After 5 days GDNF and BDNF were added to themedium (10 ng/mL).

Results

Transfection of MSCs with various miRs and miR inhibitors to inducetrans-differentiation to motor neuron progenitors and immature motorneurons was already discussed in Example 6, however, further effectivecombinations are herein described. MSCs were transfected with a controlmiR, miR-9, miR-218, miR-375, all three miRs, all three miRs and anantagomir to miR-221, and all three miRs and an antagomir to miR-373 andmRNA expression of the motor neuron markers Islet1 and HB9 was measured(FIG. 11). Expression was standardized to the control miR transfectedMSCs, which was set as 1, and the results are presented in Table 7. EachmiR on its own caused about a doubling in expression of Islet1 and HB9.Unexpectedly, expression of all three miRs had a synergistic effect ontrans-differentiation and Islet1 was increased by more than 5 fold,while HB9 was increased by more than 6 fold. The three miR combinationwas also transfected in combination with antagomirs that knockdownexpression of miR-221 or miR-373. These combinations were even moreeffective, as 8-9 fold increases in Islet1 and HB9 were observed.

TABLE 7 (Relative mRNA expression/S12 mRNA) Islet1 HB9 Con miR 1 1 miR-91.8 2.12 miR-218 2.09 1.96 miR-375 2.43 2.39 miR-9 + 218 + 375 5.23 6.483 miRs + anti-miR-221 8.6 7.9 3 miRs + anti-miR-373 9.2 8.45

Example 9 Combination RTVP-1 Silencing and miRNA Expression toDifferentiate MSCs to NSCs

Already having shown that RTVP-1 silencing alone was sufficient to driveMSC trans-differentiation to NSCs as measured by nestin mRNA expression,it was next investigated whether the silencing in combination with miRexpression could enhance the levels of nestin expressed. MSCs werecontacted with a control siRNA, RTVP-1 silencing agent alone (an siRNAagainst RTVP-1, with the sequence AAGACTGCGTTCGAATCCATA (SEQ ID NO:481), or the agent in combination with miR-218, miR-504, miR-9, miR-125or anti-miR-31 (FIG. 12). As compared to the siRNA control cells, RTVP-1knockdown alone increased nestin mRNA expression by 3.9-fold. Additionof any of the above enumerated miRs or anti-miRs further increasednestin expression; and the addition of miR-504 or miR-9 more thandoubled nestin expression. Knockdown of miR-31 in combination withRTVP-1 silencing also had a very strong synergistic effect, although notquite a doubling as compared to RTVP-1 silencing alone.

Example 10 Use of Motor Neuron-Differentiated MSCs to Treat ALS

SOD1G93A mice were used as a model for amyotrophic lateral sclerosis(ALS). Placenta-derived MSCs were trans-differentiated to motor neuronsas described in Example 8 (transfection of miR-9, miR-218 and miR-375)and these cells were administered intrathecally (5×10{circumflex over( )}5-1×106 cells) to pre-symptomatic mice (90 days old). Cells weresubsequently re-administered 1 week later. SOD1G93A mice were alsoadministered placenta-derived MSCs that had been transfected withcontrol miRs and well as administered PBS as a control. The meansurvival of eight control mice who received only PBS was 135.1. The meansurvival of eight control mice who received control MSCs was 132.5 days.Administration of motor neuron differentiated MSCs resulted in astatistically significant increase in the mean survival of the eighttest mice, as these mice survived, on average, 160.4 days.

Example 11 Use of Motor Neuron-Differentiated MSCs to Treat Spinal CordInjury

The ability of the motor neuron-differentiated MSCs to treat nerve, andspecifically spinal cord, injury was investigated. Wild-type ratsunderwent spinal cord perfusion injury by blocking the abdominal aortabelow the left renal artery for 15 minutes. The injured rats were thentreated with PBS or motor neuron-differentiated MSCs (1×10{circumflexover ( )}7 cells) injected at the L5-L6 segment of the spine. Four dayslater lower limb movement in the rats was evaluated using the Basso,Beattie and Bresnahan (BBB) locomotor scale method. Uninjured rats werealso evaluated as a control. The BBB scale is a well-established anddiscriminating method for measuring behavioral outcome and forevaluating treatments after spinal cord injury. The scale ranges fromzero to 21, with a higher score indicating superior movement. Thescoring can be summarized by the following breakdown:

0-7: Isolated joint movements with little or no hindlimb movement.

8-13: Intervals of uncoordinated stepping.

14-21: Forelimb and hindlimb coordination.

As can be seen in FIG. 13, uninjured mice had a nearly perfect score of20.6 on the BBB scale, whereas control injured mice treated with onlyPBS scored in the lowest category with an average score of 4.2. Micetreated with motor neuron-transdifferentiated MSCs showed a strongimprovement in locomotion, with an average score of 11.8, which is theupper half of the middle category, and were capable of uncoordinatedstepping.

Example 12 Use of NSC-Differentiated MSCs to Glioma

The ability of MSC transdifferentiated to NSCs by silencing of RTVP-1 totreat gliomas was investigated. Glioma stem cell (GSC)-derivedxenografts were grown in immune-compromised mice, and the mice weretreated with either RTVP-1 silenced MSCs transdifferentiated to NSCs (asdescribed in Example 9), or MSCs expressing control molecules. Thetransdifferentiated MSCs decreased tumor growth by 59.7% as compared tothe control MSC where the decrease was only 41.1%. Further, glioma cellshighly express RTVP-1, indeed its other name is gliomapathogenesis-related protein-GLIPR1, but normal healthy brain tissuedoes not. After dissection of the GSC xenografts it was found thatRTVP-1 silenced MSCs greatly reduced RTVP-1 levels in the tumor.Isolated exosomes from the transdifferentiated MSCs contained highlevels of the siRNA, suggesting that the decrease in RTVP-1 in the tumorwas likely a result of MSC-derived exosome transfer of the siRNA totumor cells.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

The invention claimed is:
 1. A method of promoting mesenchymal stem cell(MSC) differentiation toward a motor neuron cell, the method comprising:(i) culturing a MSC in a medium supporting motor neuron cell growth anddifferentiation; (ii) introducing into said MSC the following exogenousmicroRNAs (miRs): miR-9, miR-218 and miR-375; and (iii) confirmingincreased expression of at least one motor neuron marker selected fromthe group consisting of Islet1 and HB9 by detecting expression of saidmarker in said MSC, thereby promoting differentiation of the MSC intothe motor neuron cell.
 2. The method of claim 1, wherein saidintroducing comprises any one of: (i) transfecting said MSCs with anexpression vector which comprises a polynucleotide sequence whichencodes a pre-miRNA of said miR; or (ii) transfecting said MSCs with anexpression vector which comprises a polynucleotide sequence whichencodes said miR.
 3. The method of claim 1, further comprisingintroducing into said MSC a miR-221 antagomir or a miR-373 antagomir,before said confirming.
 4. A method of promoting mesenchymal stem cell(MSC) differentiation toward a motor neuron cell, the method comprising:(i) culturing a MSC in a medium supporting motor neuron cell growth anddifferentiation; (ii) introducing into said MSC the following exogenousmicroRNAs (miRs): miR-9, miR-218 and miR-375; and (iii) confirmingexpression of at least one motor neuron marker selected from the groupconsisting of Islet1 and HB9, wherein said expressing results in atleast 50% of the MSCs expressing said motor neuron marker, therebypromoting differentiation of the MSC into the motor neuron cell.
 5. Themethod of claim 4, wherein said introducing comprises any one of: (i)transfecting said MSCs with an expression vector which comprises apolynucleotide sequence which encodes a pre-miRNA of said miR; or (ii)transfecting said MSCs with an expression vector which comprises apolynucleotide sequence which encodes said miR.
 6. The method of claim4, further comprising introducing into said MSC a miR-221 antagomir or amiR-373 antagomir, before said confirming.